Method and system for providing buffer solutions

ABSTRACT

A method for delivering a processing solution includes providing a concentrated processing solution in a bioprocess container, the concentrated processing solution being produced at a first site. The method also includes combining the concentrated processing solution with a biopolymer containing solution produced in a bioreactor at a second site different from the first site. In some embodiments, the processing solution is a buffer for processing products of cells cultured in a bioreactor. A system and a pharmaceutical production facility for carrying out the method is also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 62/370,041 filed on Aug. 2, 2016, which ishereby incorporated herein by reference in its entirety for allpurposes.

FIELD OF THE INVENTION

The disclosure relates to solutions, e.g., buffers, for processingproducts of cells cultured in a bioreactor.

BACKGROUND

Bioreactors are used to produce large quantities of desired bioproducts.After growth of cells in a bioreactor to produce the bioproduct,subsequent steps are performed to isolate and concentrate thebioproduct. These processing steps can require large volumes ofexpensive processing solutions to maintain sterility and ensure that thebioproduct retains its activity. There is a need for a more costeffective way to produce solutions for processing bioproducts.

SUMMARY

An aspect of the present disclosure is related to a system fordelivering a processing solution, the system comprising a bioprocesscontainer containing a concentrated processing solution, theconcentrated processing solution produced at a first site; a processingarea having at least one processing unit at a second site different fromthe first site; and a pipe connecting the bioprocess container to the atleast one processing unit, the pipe having a valve to allow theconcentrated processing solution to flow from the bioprocess containerto the at least one processing unit to combine the concentratedprocessing solution with a biopolymer-containing solution produced inthe at least one processing unit, wherein the processing solution is atleast one of a buffer and media.

In some embodiments, the system further includes a controller connectedto the at least one processing unit, wherein the concentrated processingsolution is provided based on a determination by the controller that theprocessing solution is required when the determination is communicatedfrom the second site to the first site.

In some embodiments, the system is configured to dilute the concentratedprocessing solution prior to combining with the biopolymer containingsolution.

In some embodiments, the processing solution is Tris, Tris-Base,Tricine, HEPES, MOPS, PIPES, TAPS, bicine, BES, TES, cacodylate, MES,acetate, MKP, ADA, ACES, glycinamide, acetamidoglycine, acetic acid,citric acid, glycine, glycine glycinate, sodium phosphate, ethanol,hydrochloric acid, sodium hydroxide, guanidinium chloride, guanidinehydrochloride, sodium chloride, and/or a combination of any of these.

In some embodiments, the bioprocess container includes an inner layerfor contacting the processing solution and an outer layer configured tosupport the inner layer.

In some embodiments, the inner layer is polyethylene, and the outerlayer is a blend of polyethylene, EVOH, nylon, and PVDC.

In some embodiments the system includes a controller configured todetermine a bioburden of the processing solution.

In some embodiments, the system includes a dilution liquid supply,wherein a concentrated processing solution from the bioprocess containerand water and/or buffer from the dilution liquid supply are added to aninlet of an inline processing solution dilution system to result in adiluted processing solution.

In some embodiments, the at least one processing unit further includes aplurality of bioreactors.

In some embodiments, the system includes a plurality of bioprocesscontainers.

In some embodiments, the processing solution is a buffer.

Another aspect of the present disclosure is related to a method fordelivering a processing solution, the method comprising providing aconcentrated processing solution in a bioprocess container, theconcentrated processing solution being produced at a first site; andcombining the concentrated processing solution with abiopolymer-containing solution produced in a bioreactor at a second sitedifferent from the first site, wherein the processing solution is atleast one of a buffer and media.

In some embodiments, the concentrated processing solution is providedbased on a determination that the concentrated processing solution isrequired and the determination is communicated from the second site tothe first site.

In some embodiments, the method includes diluting the concentratedprocessing solution prior to combining with the biopolymer containingsolution.

In some embodiments, the processing solution is Tris, Tris-Base,Tricine, HEPES, MOPS, PIPES, TAPS, bicine, BES, TES, cacodylate, MES,acetate, MKP, ADA, ACES, glycinamide, acetamidoglycine, acetic acid,citric acid, glycine, glycine glycinate, sodium phosphate, ethanol,hydrochloric acid, sodium hydroxide, guanidinium chloride, guanidinehydrochloride, sodium chloride, and/or a combination of any of these.

In some embodiments, the bioprocess container comprises an inner layerfor product contact and an outer layer configured to support the innerlayer.

In some embodiments, the inner layer is polyethylene, and the outerlayer is a blend of polyethylene, EVOH, nylon, and PVDC.

In some embodiments, the method includes determining a bioburden of theprocessing solution. In some embodiments, the bioburden is determined ina rapid detection assay.

In some embodiments, the method includes adding concentrated processingsolution from the bioprocess container and water and/or buffer from adilution liquid supply to an inlet of an inline processing solutiondilution system to result in a diluted processing solution.

In some embodiments, the method includes transporting the bioprocesscontainer containing the processing solution from the first site to thesecond site.

In some embodiments, the method includes feeding bioprocessing solutionthrough an outlet in the bioprocess container to facilitate purificationof the biopolymer.

Another aspect of the present disclosure is related to a pharmaceuticalproduction facility comprising a buffer storage area capable ofreceiving at least one bioprocess container configured to contain aconcentrated processing solution; a processing area having at least oneprocessing unit; a wall separating the buffer storage area from theprocessing area; and at least one pipe having a first end in the bufferstorage area and a second end in the processing area, the first endconfigured to connect to at least one bioprocess container of the atleast one bioprocess container, and the second end configured to connectto at least one processing unit of the at least one processing unit.

In some embodiments, the at least one pipe has a valve.

In some embodiments, the at least one bioprocess container includes aninner layer for contacting the processing solution and an outer layerconfigured to support the inner layer.

In some embodiments, the pharmaceutical production facility of claim,further includes a platform in the buffer storage area, the platformbeing configured to support the at least one bioprocess container, theat least one bioprocess container including a plurality of bioprocesscontainers, the platform having a first level configured to support atleast one bioprocess container of the plurality of bioprocess containersand a second level configured to support at least one bioprocesscontainer of the plurality of bioprocess containers, wherein theplatform is configured to allow a user to place a bioprocess containerof the plurality of bioprocess containers onto the platform using aforklift, and the platform being configured to allow a user to removethe respective bioprocess container from the platform using theforklift.

In some embodiments, the pharmaceutical production facility furtherincludes a plurality of inlets spaced apart along the platform, eachinlet being connected to the at least one pipe, and each inlet beingconfigured to be connected to an outlet of a bioprocess container of theplurality of bioprocess containers.

In some embodiments, the processing area is configured as a cleanroomenvironment.

In one aspect, the invention provides a method, e.g., a method ofdelivering a bioreactor processing solution or a method of forming abiopolymer-containing solution. The method includes providing (e.g.,receiving) a concentrated processing solution; combining theconcentrated processing solution with a biopolymer-containing solutionproduced in a bioreactor, e.g., to form a biopolymer-containingsolution.

In embodiments, the concentrated processing solution is provided in aunit of at least 20 liters, of about 20 to 2000 liters, e.g., 75, 100,125, 250, 500, 1000, 1250, 1500, 1750, 1825, 1900, or 2000 liters.

In embodiments, the biopolymer containing solution comprises abiopolymer from tables entitled “Therapeutic Products and “ExemplaryProducts, e.g., Bispecific Molecules”, below.

In embodiments, the biopolymer is made from a Chinese hamster ovary(CHO) cell. In one embodiment, the cell is a CHO-K1 cell, a CHO-K1 SVcell, a DG44 CHO cell, a DUXB11 CHO cell, a CHOS, a CHO GS knock-outcell, a CHO FUT8 GS knock-out cell, a CHOZN, or a CHO-derived cell. TheCHO GS knock-out cell (e.g., GSKO cell) is, for example, a CHO-K1SV GS.

In embodiments, the processing solution is provided in one or morebioprocess containers.

In embodiments, the processing solution is provided in one or morebioprocess containers and is used for a single dilution.

In embodiments, the method comprises providing a value for theparameter, and optionally, comparing the value to a reference.

In embodiments, the evaluation is performed at a first site and thebioreactor is located at a second site.

In embodiments, the evaluation is performed at the site of thebioreactor.

In embodiments, the method comprises diluting the concentratedprocessing solution prior to combining with the biopolymer containingsolution.

In embodiments, the processing solution is shipped or produced at afirst site and the bioreactor is located at a second site.

In embodiments, the processing solution comprises a buffer, e.g., Tris,Tricine, HEPES, MOPS, PIPES, TAPS, bicine, BES, TES, cacodylate, MES,acetate, MKP, ADA, ACES, glycinamide and acetamidoglycine or acetic acidor similar.

In embodiments, the bioprocess container comprises an inner layer forproduct contact and an outer layer.

In embodiments, the inner layer is polyethylene.

In embodiments, the outer layer is a blend of polyethylene, EVOH, nylon,and/or PVDC.

In some embodiments, the method includes determining the bioburden ofthe processing solution.

In embodiments, the method comprises adding concentrated bioprocessingsolution (concentrated processing solution) from a processing solutionsupply and water or buffer from the water/buffer supply to an inlet ofan inline bioprocessing solution dilution system to result in dilutedbioprocessing solution.

In embodiments, the method comprises feeding bioprocessing solutionthrough an outlet in the bioprocess container to facilitate purificationof the biopolymer.

Also provided by the invention is a system for delivering abioprocessing solution. The system includes providing a concentratedprocessing solution produced at a first site and combining theconcentrated biopolymer-containing solution with a biopolymer-containingsolution produced in a bioreactor at a second site.

In embodiments, the concentrated bioprocessing solution is providedbased on a determination that it is required and the determination iscommunicated from the second site to the first site.

Among the advantages of the invention is the ability to decoupleproduction of a downstream processing solution from the manufacture of abiologic in a bioreactor. This, in combination of the use of bufferconcentrates stored in bioprocess containers (“BPC”s aka buffer bags, ortotes) allows for production of process intermediates to be prepared ata location remote to the biologics production. This reducesmanufacturing costs. The extended stability time allows for review andrelease of GMP paperwork. As such, in process intermediates impacted bya GMP error or failed quality test can be rejected, therefore removingrisk to the biologics production.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of a prior art arrangement of a bioreactorpreparation area;

FIG. 2 is a schematic view of an embodiment of a buffer preparationarrangement according to the present disclosure;

FIG. 3 is a block diagram of an embodiment of a method according to thepresent disclosure;

FIG. 4A shows a perspective view of an embodiment of an outer layer of abioprocess container;

FIG. 4B shows a side view thereof;

FIG. 4C shows a rear view thereof;

FIG. 4D shows a side view thereof;

FIG. 4E shows a front view thereof;

FIG. 4F shows a top view thereof;

FIG. 5 shows a side view of another embodiment of an outer layer of abioprocess container;

FIG. 6A shows a front view of an embodiment of a platform;

FIG. 6B shows a side view thereof;

FIG. 7A shows a side view of an embodiment of a buffer storage area in apharmaceutical production facility;

FIG. 7B shows a front view thereof; and

FIG. 7C shows a top view thereof.

DETAILED DESCRIPTION

The invention provides cost-effective processing solutions forconcentrating and purifying biologic products of large scale expressionvessels, including bioreactors.

Biologics manufacturing typically include two main manufacturing stages.The first is a biologic product production stage, in which a biologic,typically a biopolymer such as a polypeptide or protein, is produced.The biopolymers are typically synthesized using microbial fermentation,biotransformation or mammalian cell culture. Bioreactor expressionsystems are disclosed in, e.g., U.S. 2011-0312087 A1.

The second step includes purifying the biologic product, in which theproduct is separated from impurities e.g. cellular debris, unwantedbiopolymers, DNA media components and catabolytes. The second step canrequire large volumes of a biocompatible solution that preserves to theextent possible the desired activity of the biologic and is otherwisebiocompatible with the biologic in order to selectively removeimpurities a number of dowstream processing stages (unit operations) areexecuted. During these unit operations a number of technologies are usedincluding size exclusion filtration, chromatography, pH inactivation,refolding, tangential flow filtration, etc. To facilitate these unitoperations a number of different aqueous solutions (buffers) are usedwhich alter the physicochemical environment of the target molecule (e.gpH, conductivity).

A prior art arrangement of a bioreactor preparation area 10 is shown inFIG. 1. The buffer preparation area having a buffer prepration vessel 12is in close physical proximity to a buffer storage area having a bufferhold tank 14 and a portable tote 16. The buffer preparation vessel 12 ofthe buffer preparation area is also in close physical proximity to adownstream processing area having a downstream processing unit, such asa chromatography column 18. A pipe 20 connects the buffer preparationvessel 12 to the buffer hold tank 14 and the portable tote 16. Anotherpipe 22 connects the buffer hold tank 14 to the downstream processingunit 18 in the downstream processing area. Yet another pipe 24 connectsthe portable tote 16 to the downstream processing unit 18 in thedownstream processing area. This arrangement can be expensive and timeconsuming, which results in in-process material being used prior tocompletion of long lead testing and/or review and release of GMP records(batch records). Stockpiling of buffers occupies a large amount of GMPprocessing space.

In contrast, FIG. 2 shows an embodiment of a system 100 for delivering aprocessing solution according to the present disclosure. As shown, thesystem 100 includes a buffer preparation area 102 at first site 104,which is distinct from a downstream processing area 106 at a second site108. In some embodiments, the first site 104 is a campus or building andthe second site 108 is a campus or building distinct from the campus orbuilding of the first site 104.

While the buffer preparation area 102 and the downstream processing area106 may be within one larger area, e.g., a factory or production site,no proximity is required as long as the concentrated solutions can bedelivered to the downstream processing area without adversely affectingthe stability or activity of the solution. Scheduling, formulation, anddelivery of the solutions can be performed to prepare the solutions ator just before their intended time of use.

In addition, the arrangement according to the disclosure allows fordownstream processing solutions to be performed at a low value location,which can be preferable to preparing them at a high value locationincluding the bioreactor. The arrangement also reduces storage space atthe site of the bioreactor.

In FIG. 2, the buffer preparation area 102 includes at least two bufferpreparation vessels, each indicated at 110. The buffer preparationvessels 110 are connected by at least one pipe (or at least onemanifold) 112 to several storage tanks (or totes), each indicated at114, in a buffer storage area 116. The buffer storage area 116 alsoincludes at least one tote 114 that has been filled and is disconnectedfrom the manifold so that it is ready to be transported to the secondsite 108.

The buffer preparation vessels 110 are useful for producing a processingsolution. In some embodiments, the processing solution is a buffer suchas Tris, Tricine, HEPES, MOPS, PIPES, TAPS, bicine, BES, TES,cacodylate, MES, acetate, MKP, ADA, ACES, glycinamide andacetamidoglycine or acetic acid or a buffer as shown in Tables 6-9.

Each tote 114 includes an inner layer or liner 118 for contacting theconcentrated processing solution and an outer layer or body 120 thatsupports the inner layer. In some embodiments, the inner layer 118 ismade of polyethylene. In some embodiments, the outer layer 120 is ablend of polyethylene, ethylene vinyl alcohol (EVOH), nylon, andpolyvinylidene chloride (PVDC). In some embodiments, each tote 114 ismade of plastic. In some embodiments, each tote 114 is capable of beingsterilized by gamma radiation.

In some embodiments, each tote 114 is configured to ensure a shelf lifeof the processing solution contained within the tote for one month. Insome embodiments, each tote 114 is configured to ensure a shelf life ofthe processing solution contained within the tote for three months. Insome embodiments, each tote 114 is configured to ensure a shelf life ofthe processing solution contained within the tote for six months. Insome embodiments, each tote 114 is configured to ensure a shelf life ofthe processing solution contained within the tote for one year.

In some embodiment each tote 114 has a volume of 1,000 liters (L). Insome embodiments, each tote 114 has a rigid outer layer 120 that iscapable of retaining a defined shape without external support on sidewalls of the tote 114.

At the buffer storage area 116, a user places one or more totes 114 on ashipping structure, such as a pallet. In one embodiment, a packaged totesupported on a pallet forms a shipping unit 122 is a 4 foot×4 foot×4foot cube. The user places one or more shipping units 122 into a vehicle124, such as a truck. The user drives the vehicle 124 from the bufferstorage area 116 at a first facility (first site) 104 along the arrow Ato a second facility (second site) 108 housing the downstream processingarea 106.

At the second facility 108, the user unloads the shipping unit(s) 122from the vehicle 124 along the arrow B. Then the user places the tote(s)114 in a second buffer storage area 126 in the second site 108.

The second facility 108 includes at least one bioreactor 128 located indownstream processing area 130. In some embodiments, the downstreamprocessing area 130 is a cleanroom. In the shown embodiment, two pipes,each indicated at 132, connect the totes (bioprocess containers) 114located in the second buffer storage area 126 to the bioreactor 128located in the downstream processing area 130. As shown, each pipe 132includes a valve 134 that is positioned within or adjacent to a wall 136of the downstream processing area 130, which separates the second bufferstorage area 126 from the downstream processing area 130. The pipes 132and the valves 134 in or adjacent the wall 136 allow the concentratedprocessing solution to flow from the bioprocess containers 114 to thebioreactor 128 to combine the concentrated processing solution with abiopolymer-containing solution produced in the bioreactor 128.

As mentioned above, the wall 136 separates the downstream processingarea 130, which is formed as a cleanroom, from the second storage area126, which is not necessarily a cleanroom. The valve 134 allows for acleanroom compliant connection of the one or more totes 114 to the valveto minimize the risk of contaminating the bioreactor 128.

In some embodiments, each valve 134 depends from a ceiling at the secondsite 108.

When the processing solution is needed in the downstream processing area106, the processing solution is pumped or otherwise moved from the totes114 through the pipes 132 to the downstream processing unit 128 in thedownstream processing area 106.

In some embodiments, the second site 108 includes a plurality ofbioreactors 128 (or other downstream processing units) that areconnectable to one or more totes 114. In some embodiments, only one tote114 is used.

In some embodiments, the second site 108 includes at least one tote 114that is currently connected to one or more bioreactors 128 as well as atleast one tote 114 that is not yet connected to one or more bioreactors128. This ensures that the user of the second site 104 has a backupsupply of the processing solution contained in the totes 114.

The bioreactor 128 is connected to a controller 140. In someembodiments, the controller 140 is configured to determine that theprocessing solution contained in one or more totes 114 is required bythe one or more bioreactors 128 at the second site 108. The controller140 then communicates this determination to a controller 142 in thefirst site 104. This communication of the determination prevents ashortage of the processing solution at the second site 108.

In some embodiments, the controller 140 is configured to determine thebioburden of the processing solution. For example, the bioburden can bedetermined in a rapid detection assay. In some embodiments, thebioburden is determined in a Raman spectroscopy rapid detection assay.

The system 100 is configured to dilute the concentrated processingsolution prior to combining with the biopolymer containing solution.This allows the system 100 to adjust the parameters such as the pH ofthe solution that is provided to the bioreactor 128. The system 100includes a processing solution supply in the form of totes 114. Thesystem 100 also includes a dilution liquid supply 144. In someembodiments, the dilution liquid can be water and/or a buffer. Theconcentrated processing solution from the totes 114 and water or bufferfrom the dilution liquid supply 144 are added to an inlet of an inlineprocessing solution dilution system 146 along the pipes 132 to result ina diluted processing solution that is provided at an inlet of thebioreactor 128.

FIG. 2 shows only one bioreactor 128. More bioreactors may be includedat the second site 108 without departing from the scope of the presentdisclosure. In some embodiments, the bioreactor(s) 128 in the secondsite 108 are useful for purification. In some embodiments, thebioreactor(s) 128 in the second site 108 include at least onechromatography column.

Referring now to FIG. 3, the present disclosure provides a method 300for delivering a processing solution, the system. In FIG. 3, anexemplary embodiment of the method 300 for delivering a processingsolution includes producing a concentrated processing solution at afirst site at 302. In some embodiments, the processing solution is abuffer.

In some embodiments, the buffer is Tris, Tricine, HEPES, MOPS, PIPES,TAPS, bicine, BES, TES, cacodylate, MES, acetate, MKP, ADA, ACES,glycinamide and acetamidoglycine or acetic acid or a buffer as shown inTables 6-9.

In some embodiments, at 304, the method 300 determines the bioburden ofthe processing solution. Step 304 can be performed entirely or in partby a controller, such as the controller 140 in FIG. 2. At step 304, themethod 300 determines that the processing solution meets qualitystandards. This allows a user to reduce the risk that a product from thebioreactor (or other downstream processing unit) will be contaminated.Any bioburden testing method can be implemented. In some embodiments,the bioburden can be determined in a rapid detection assay. In someembodiments, the bioburden is determined in a Raman spectroscopy rapiddetection assay. In some embodiments, the bioburden testing can be anin-line or at-line test performed at either the processing solutionproduction site or a second site where the processing solution is to beused.

At 306, the method 300 determines that the concentrated processingsolution is required at the second site. At 308, the method communicatesthis determination from the second site to the first site. Thisdetermination at step 306 can be performed by a controller connected toa bioreactor at the second site, such as the controller 140 shown inFIG. 2. The concentrated processing solution is then provided to thesecond site 108 based on the determination.

At 310, the method 300 provides a concentrated processing solution in abioprocess container, the concentrated processing solution produced at afirst site. In some embodiments, step 310 includes transporting one ormore bioprocess containers containing the processing solution from thefirst site to the second site.

In some embodiments, the bioprocess container includes an inner layerfor contacting the process solution and an outer layer. In someembodiments, the inner layer is polyethylene. In some embodiments, theouter layer is a blend of polyethylene, EVOH, nylon, and PVDC.

At 312, the method 300 includes diluting the concentrated processingsolution prior to combining with the biopolymer containing solution. Forexample, in some embodiments, step 312 of the method 300 includes addingconcentrated processing solution from a processing solution supply andwater or buffer from the water/buffer supply to an inlet of an inlineprocessing solution dilution system to result in a diluted processingsolution.

At 314, the method 300 combines the concentrated processing solutionwith a biopolymer-containing solution produced in a bioreactor at thesecond site, which is different from the first site.

The compositions, systems, and methods of the disclosure provideextended stability data and allow for long term storage of processintermediates. This allows an operator to decouple production of abiologic product from production of an intermediate used to process thebiologic product. A further advantage of the disclosure is that itallows for a concentrated buffer solution to be produced at a siteremote from the site of biologic production. The concentrated bufferscan be produced in a low value production facility and thus frees upspace in a high value production facility occupied by the bioreactor. Inaddition to reducing manufacturing costs, the extended stability timeallows for review and release of GMP paperwork. Process intermediatesimpacted by a GMP error or failed quality test etc. can be rejected,thus removing risk to the biologics production.

The combined use of concentrated buffer supplied in BPCs from a remotepreparation area, in line dilution and long expiry times for the buffersolutions results in a cheaper and less risk laden buffer supplystrategy.

The systems and methods of the present disclosure can use variousbioprocess containers. FIGS. 4A-4F show an outer layer generallyindicated at 400 of an embodiment of a bioprocess container. The outerlayer 400 can be used to support an inner layer, such as a bag, forcontaining a buffer within the inner layer. The lower end of the outerlayer 400 of the bioprocess container includes a portion that can belifted by a forklift. FIG. 4A shows a perspective view of the bioprocesscontainer.

In FIG. 4B, the bioprocess container has an overall height C of 1264millimeters. FIG. 4C shows another side of the bioprocess containerhaving a height D of 1287 millimeters when a lid is secured to the outerlayer 400 of the bioprocess container.

The lower end of the bioprocess container can be engaged by a forkliftand includes base portions 402, 404, 406 that define recesses 408, 410.Each of the recesses 408, 410 has a height E of 135 millimeters. A firstrecess 408 has a width F of 310 millimeters. A second recess 410 has awidth H of 310 millimeters. A first base portion 402 has a width G of160 millimeters. A second base portion 404 has a width I of 180millimeters.

In FIG. 4F, the top view of the outer layer 400 of the bioprocesscontainer has a length J of 1140 millimeters and a width K of 1140millimeters. The inner volume of the outer layer 400 of the bioprocesscontainer is defined by inner length L and inner width M. The innerlength L is 1056 millimeters and the inner width M is 1038 millimeters.

A port 412 is defined in the outer layer 400 of the bioprocess containerfor dispensing process solution from the bioprocess container. The port412 has a diameter R of 97 millimeters.

The port 412 is offset from a first inner surface 414 of the outer layer400 of the bioprocess container by dimension Q, which is 171millimeters. The port 412 is offset from a second inner surface 416 ofthe outer layer 400 of the bioprocess container by dimension S, which is181 millimeters. The port 412 is offset from a first outer surface 418by dimension P, which is 222 millimeters. The port 412 is offset from asecond outer surface 420 of the by dimension O, which is 222millimeters.

In some embodiments, the inner layer (bag) of the bioprocess containerincludes outlet tubing that is threaded through the port 412 of theouter layer 400.

FIG. 5 shows another embodiment of an outer layer generally indicated at500 of a bioprocess container. In FIG. 5, the outer layer 500 of thebioproces sing unit has a height U of 593 millimeters and a width V of1140 millimeters. The height T of the outer layer 500 of thebioprocessing unit and the lid secured to the outer layer 500 is 617millimeters.

In some embodiments, the bioprocess container includes a trackingsystem. For example, in some embodiments, the bioprocess containerincludes an RFID tag 422 (FIG. 4A) affixed to the outer layer 400 of thebioprocess container to facilitate tracking and management of thebioprocess container during shipping receiving. By including arespective RFID tag 422 on multiple bioprocess containers, a user canmore easily manage inventory of bioprocess containers and more easilymonitor the quality of the processing solution contained in thebioprocess container.

FIG. 6A shows a front view of an embodiment of a platform generallyindicated at 600 for supporting bioprocess containers 602 to organize abuffer storage area 604. The platform 600 is configured to support atleast one bioprocess container 602. This platform 600 can be used tosupport bioprocess containers in other embodiments of the buffer storagearea, such as buffer storage area 126 of FIG. 2.

The platform 600 has a first level 606 configured to support at leastone bioprocess container 602 and a second level 608 configured tosupport at least one bioprocess container 602. The platform 600 isconfigured to allow a user to place a bioprocess container 602 onto theplatform 600 using a forklift, and the platform 600 is configured toallow a user to remove the respective bioprocess container 602 from theplatform 600 using the forklift.

The first level 606 is offset from the ground by height AA, which is 2feet. The platform is supported by feet, each indicated at 610, thathave a height AB of 2 inches. Columns, each indicated at 612, extendupwardly from the feet 610, and support the first level 606 and thesecond level 608.

In FIG. 6A, a lower portion of a bioprocess container 602 includes threebase portions 614, 616, 618 that define two recesses 620, 622. Therecesses 620, 622 allow a user to operate a forklift to lift eachbioprocess container 602 by accessing the respective bioprocesscontainer 602 from the front of the platform 600.

The recesses 620, 622 have a height AC of 5 and 5/16 inches. The firstbase portion 614 has a width AD of 7 and ⅛ inches. The second baseportion 616 is offset from a side of the bioprocess container 602 by adistance AE of one foot and 7 and 5/16 inches. The second base portion616 has a width AF of 6 and ¼ inches. The third base portion 618 isoffset from a side of the bioprocess container 602 by a distance AG of 3feet and 1 and ¾ inches. The third base portion 618 has a width AH of 7and ⅛ inches.

Each bioprocess container 602 has a height AI of 4 feet and 3 inches anda width AJ of 3 feet and 8 and ⅞ inches. The clearance AK between thebioprocess container 602 on the second level 608 of the platform 600 andthe ceiling of the buffer storage area 604 is 6.5 inches. The overallheight AL of the upper edge 626 of the bioprocess container 602 on thesecond level 608 of the platform 600 is 11 feet and five inches from theground 628. The ceiling 624 is a height AM of 11 feet and 11.5 inchesabove the ground 628.

The bioprocess container 602 on the first level 606 is positionedbetween the columns 612 so there is a clearance AN of 3 inches betweenthe bioprocess container 602 and at least one of the columns 612. Thereis a clearance AO of 6 and ⅜ inches between the lower surface 630 of thesecond level 608 and the upper surface 632 of the bioprocess container602 that is positioned on the first level 606.

FIG. 6B shows a side view of the platform 600 of FIG. 6A. FIG. 6B showsthat the bioprocess container has a depth AP of 3 feet and 8⅞ inches.

An aspect of the present disclosure relates to a pharmaceuticalproduction facility having a buffer storage area capable of receiving atleast one bioprocess container configured to contain a concentratedprocessing solution, a processing area capable of receiving at least oneprocessing unit, and a wall separating the buffer storage area from theprocessing area. The facility also includes at least one pipe having afirst end in the buffer storage area and a second end in the processingarea. The first end is configured to connect to at least one bioprocesscontainer, and the second end is configured to connect to at least oneprocessing unit. In some embodiments, the processing area is configuredas a cleanroom environment.

FIG. 7A shows a buffer storage area generally indicated at 700 that canbe included in a pharmaceutical production facility. The buffer storagearea 700 includes a platform 702 for supporting a plurality ofbioprocess containers 704.

Each bioprocess container 704 includes an inner layer for contacting theprocessing solution and an outer layer configured to support the innerlayer. Each bioprocess container 704 can be a bioprocess container ofany of the embodiments shown and described herein.

FIG. 7A shows how a user 706 can access the bioprocess containers 704from a user walkway 708 adjacent to the platform.

The pharmaceutical facility includes at least one pipe having a firstend in the buffer storage area 700 and a second end in the processingarea, such as a processing area as described in relation to variousembodiments herein. The first end of each pipe is configured to connectto at least one bioprocess container 704, and the second end isconfigured to connect to at least one processing unit in the processingarea. As described in relation to FIG. 2, each pipe can include a valve.

FIG. 7B shows that the buffer storage area 700 includes a plurality ofinlets, each indicated at 710 spaced apart along a length of theplatform 702. Each inlet 710 is connected to the pipe(s). Each inlet 710is configured to be connected to an outlet of a bioprocess container.The inlets 710 and the pipe(s) allow buffer to flow from one or more ofthe bioprocess container(s) 704 to a processing unit in the processingarea.

Referring back additionally to FIG. 7A, the platform 702 supportsbioprocess containers 704 on a first level 712 of the platform 702 andon a second level 714 of the platform between a floor 716 and a ceiling718 of the buffer storage area 700. The platform 702 allows a user 706to organize the bioprocess containers 704 in a way that efficiently usesthe space of the buffer storage area 700 and that allows the user toplace the bioprocess containers 704 on the platform 702 and remove thebioprocess containers 704 from the platform 702 using a forklift.

In embodiments in which the bioprocess containers 704 include a trackingsystem, such as a barcode or an RFID tag affixed to each bioprocesscontainer 704, the user walkway 708 allows a user to easily access thebioprocessing units to take inventory of the bioprocess containers 704or to collect information such as the age of the bioprocess containers704.

The user walkway 708 also allows a user 706 to connect a port on one ofthe bioprocess containers 704 to an inlet 710 so that process solutionfrom that bioprocess container 704 can flow from the bioprocesscontainer 704 through the inlet 710 and the pipe to one or moreprocessing unit(s) in the processing area.

The top view of FIG. 7C shows that the walkway 708 has a width BA of 3feet.

The walkway 708 is connected to two stairways 720, 722. The walkway 708and the first stairway 720 each has a width BA of 3 feet. The secondstairway 722 has a width BB of 2.5 feet. The walkway 708 is positioned adistance BC1 of 6 inches from a rear wall 724 and a distance BC2 of 6inches from each platform 702. The walkway 708 is positioned a distanceBD of 1 foot and 9 inches from a side wall 726.

The platform 702 includes feet, each indicated at 728. The foot 728closest to the side wall 726 is centered a distance BE of 2 feet and1.25 inches from the side wall 726.

The inlets 710 are spaced apart, with a first inlet 710 being a distanceBF of 10.75 feet from the side wall 726, a second inlet 710 being adistance BG of 10.5 feet from the first inlet 710, a third inlet being adistance BH of 9 feet five inches from the second inlet 710, a fourthinlet 710 being a distance BI of 10 feet from the third inlet 710, and afifth inlet 710 being a distance BJ of 10 feet 2 inches from the fourthinlet 710.

Two platforms 702 are included in the buffer storage area 700. Eachplatform 702 includes frame members 730 having a cross section of 3inches wide and 2 inches high. The platforms 702 are spaced apart by adistance BK of 6 feet and ⅝ inches

Building support columns 732, 734, 736 extend between the floor 716 andthe ceiling 718. The distance BL between the first support column 732and the second support column 734 is 27 feet and 2.5 inches. Thedistance BM between the second support column 734 and the third supportcolumn 736 is 27 feet and 2.5 inches. The width BN of the second supportcolumn 734 is 3 feet and 10 inches. A face 738 of the second supportcolumn is a distance BO of 6 feet and 7 inches from the front edge ofthe platforms 702.

The dimensions discussed in relation to FIGS. 4A-7C are exemplary andcan be changed to suit a particular facility.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice of and/or for the testing of the present disclosure, thepreferred materials and methods are described herein. In describing andclaiming the present disclosure, the following terminology will be usedaccording to how it is defined, where a definition is provided.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “a cell” can mean one cell or more than onecell.

As used herein, “processing solution” means a buffer or media used in abiologic manufacturing process including solutions used in upstream anddownstream processing. As used herein a downstream processing agent(DPA) is a biocompatible solution that contains one or more ingredientsthat act upon a product of a bioreactor or associated component, e.g., acell cultured in a bioreactor, for downstream processing operationsperformed on the target molecule. Examples of suitable DPAs include,e.g., buffered saline solution such as HBSS, PBS, TRIS, Acetic acid orsimilar. A DPA as used herein is thus distinct from a medium, which doessupport growth and division of cells. The DPAs of the disclosure providea physiologically equivalent physicochemical environment withoutsupporting cell growth and/or division.

DPAs are commonly used in downstream processing for biologicsmanufacture and heretofore are produced in a GMP manner temporally closecoupled to a production batch. In addition to adding time and cost, thisresults in in-process material being used prior to completion of longlead testing and/or review and release of GMP records (batch records) assuch in process material is used “at risk”. Using unreleased materialrisks the possibility that a GMP error could be uncovered after the useof the process intermediate that would introduce risk to productquality, these can include instrument calibration errors and the like.This method is used because buffers can have assigned use by dates ofdays and stockpiling buffers occupies a large amount of GMP processingspace.

As used herein, the term “bioburden” refers to the level ofself-replicating biological contaminants present in a composition (e.g.,solid or liquid) and/or on the surface (e.g., exterior and/or interiorsurface) of an article(s). For example, bioburden can refer toself-replicating biological contaminants present in a compositioncontaining a chromat the term “bioburden” is art known and refers to thelevel of self-replicating biological contaminants present in acomposition (e.g., solid or liquid) and/or on the surface (e.g.,exterior and/or interior surface) of an article(s). In other examples,bioburden can to refer to self-replicating biological contaminants onthe inner surface of a chromatography column and/or within thechromatography resin within the chromatography column (e.g., biologicalcontaminants on the inner surface of a chromatography column andbiological contaminants in the packed chromatography resin within thechromatography column). Bioburden can also refer to the self-replicatingbiological contaminants present within a liquid (e.g., a buffer used inany of the methods or processes described herein). Non-limiting examplesof self-replicating biological contaminants can be bacteria (e.g.,Gram-positive or Gram-negative bacteria, or a bacterial spore),mycobacteria, viruses (e.g., a vesivirus, a Cache Valley virus, aparvovirus, a herpes virus, and a bunyavirus), parasites, fungi, yeast,and protozoa.

As used herein, the term “endogenous” refers to any material from ornaturally produced inside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedto or produced outside of an organism, cell, tissue or system.Accordingly, “exogenous nucleic acid” refers to a nucleic acid that isintroduced to or produced outside of an organism, cell, tissue orsystem. In an embodiment, sequences of the exogenous nucleic acid arenot naturally produced, or cannot be naturally found, inside theorganism, cell, tissue, or system that the exogenous nucleic acid isintroduced into. In one embodiment, the sequences of the exogenousnucleic acids are non-naturally occurring sequences, or encodenon-naturally occurring products.

As used herein, the term “heterologous” refers to any material from onespecies, when introduced to an organism, cell, tissue or system from adifferent species.

As used herein, the terms “nucleic acid,” “polynucleotide,” or “nucleicacid molecule” are used interchangeably and refers to deoxyribonucleicacid (DNA) or ribonucleic acid (RNA), or a combination of a DNA or RNAthereof, and polymers thereof in either single- or double-stranded form.The term “nucleic acid” includes, but is not limited to, a gene, cDNA,or an mRNA. In one embodiment, the nucleic acid molecule is synthetic(e.g., chemically synthesized or artificial) or recombinant. Unlessspecifically limited, the term encompasses molecules containinganalogues or derivatives of natural nucleotides that have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally or non-naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions), alleles, orthologs, SNPs, andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka etal., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol.Cell. Probes 8:91-98 (1994)).

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused to refer to a compound comprised of amino acid residues covalentlylinked by peptide bonds, or by means other than peptide bonds. A proteinor peptide must contain at least two amino acids, and no limitation isplaced on the maximum number of amino acids that can comprise aprotein's or peptide's sequence. In one embodiment, a protein maycomprise of more than one, e.g., two, three, four, five, or more,polypeptides, in which each polypeptide is associated to another byeither covalent or non-covalent bonds/interactions. Polypeptides includeany peptide or protein comprising two or more amino acids joined to eachother by peptide bonds or by means other than peptide bonds. As usedherein, the term refers to both short chains, which also commonly arereferred to in the art as peptides, oligopeptides and oligomers, forexample, and to longer chains, which generally are referred to in theart as proteins, of which there are many types. “Polypeptides” include,for example, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others.

As used herein, “product” refers to a molecule, e.g., a protein, nucleicacid, polypeptide, or any hybrid thereof, that is produced, e.g.,expressed, by a cell which has been modified or engineered to producethe product. In one embodiment, the product is a naturally occurringproduct or a non-naturally occurring product, e.g., a synthetic product.In one embodiment, a portion of the product is naturally occurring,while another portion of the product is non-naturally occurring. In oneembodiment, the product is a polypeptide, e.g., a recombinantpolypeptide. In one embodiment, the product is suitable for diagnosticor pre-clinical use. In another embodiment, the product is suitable fortherapeutic use, e.g., for treatment of a disease. In one embodiment,the product is selected from Table 1 or Table 2. In one embodiment, themodified or engineered cells comprise an exogenous nucleic acid thatcontrols expression or encodes the product. In other embodiments, themodified or engineered cells comprise other molecules, e.g., that arenot nucleic acids, that controls the expression or construction of theproduct in the cell.

In one embodiment, the modification of the cell comprises theintroduction of an exogenous nucleic acid comprising a nucleic acidsequence that controls or alters, e.g., increases, the expression of anendogenous nucleic acid sequence, e.g., endogenous gene. In suchembodiments, the modified cell produces an endogenous polypeptideproduct that is naturally or endogenously expressed by the cell, but themodification increases the production of the product and/or the qualityof the product as compared to an unmodified cell, e.g., as compared toendogenous production or quality of the polypeptide.

In another embodiment, the modification of the cell comprises theintroduction of an exogenous nucleic acid encoding a recombinantpolypeptide as described herein. In such embodiments, the modified cellproduces a recombinant polypeptide product that can be naturallyoccurring or non-naturally occurring. In such embodiments, the modifiedcell produces a recombinant polypeptide product that can also beendogenously expressed by the cell or not. In embodiments where therecombinant polypeptide product is also endogenously expressed by thecell, the modification increases the production of the product and/orthe quality of the product as compared to an unmodified cell, e.g., ascompared to endogenous production or quality of the polypeptide.

As used herein, “recombinant polypeptide” or “recombinant protein”refers to a polypeptide that can be produced by a cell described herein.A recombinant polypeptide is one for which at least one nucleotide ofthe sequence encoding the polypeptide, or at least one nucleotide of asequence which controls the expression of the polypeptide, was formed bygenetic engineering (of the cell or of a precursor cell). E.g., at leastone nucleotide was altered, e.g., it was introduced into the cell or itis the product of a genetically engineered rearrangement. In anembodiment, the sequence of a recombinant polypeptide does not differfrom a naturally occurring isoform of the polypeptide or protein. In anembodiment, the amino acid sequence of the recombinant polypeptidediffers from the sequence of a naturally occurring isoform of thepolypeptide or protein. In an embodiment, the recombinant polypeptideand the cell are from the same species. In an embodiment, therecombinant polypeptide is endogenous to the cell, in other words, thecell is from a first species and the recombinant polypeptide is nativeto that first species. In an embodiment, the amino acid sequence of therecombinant polypeptide is the same as or is substantially the same as,or differs by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%from, a polypeptide encoded by the endogenous genome of the cell. In anembodiment, the recombinant polypeptide and the cell are from differentspecies, e.g., the recombinant polypeptide is a human polypeptide andthe cell is a non-human, e.g., a rodent, e.g., a CHO, or an insect cell.In an embodiment, the recombinant polypeptide is exogenous to the cell,in other words, the cell is from a first species and the recombinantpolypeptide is from a second species. In one embodiment, the polypeptideis a synthetic polypeptide. In one embodiment, the polypeptide isderived from a non-naturally occurring source. In an embodiment, therecombinant polypeptide is a human polypeptide or protein which does notdiffer in amino acid sequence from a naturally occurring isoform of thehuman polypeptide or protein. In an embodiment, the recombinantpolypeptide differs from a naturally occurring isoform of the humanpolypeptide or protein at no more than 1, 2, 3, 4, 5, 10, 15 or 20 aminoacid residues. In an embodiment, the recombinant polypeptide differsfrom a naturally occurring isoform of the human polypeptide by no morethan 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15% of its amino acid residues.

“Acquire” or “acquiring” as the terms are used herein, refer toobtaining possession of a physical entity, or a value, e.g., a numericalvalue, by “directly acquiring” or “indirectly acquiring” the physicalentity or value. “Directly acquiring” means performing a process (e.g.,performing a synthetic or analytical method) to obtain the physicalentity or value. “Indirectly acquiring” refers to receiving the physicalentity or value from another party or source (e.g., a third partylaboratory that directly acquired the physical entity or value).Directly acquiring a physical entity includes performing a process thatincludes a physical change in a physical substance, e.g., a startingmaterial. Exemplary changes include making a physical entity from two ormore starting materials, shearing or fragmenting a substance, separatingor purifying a substance, combining two or more separate entities into amixture, performing a chemical reaction that includes breaking orforming a covalent or non-covalent bond. Directly acquiring a valueincludes performing a process that includes a physical change in asample or another substance, e.g., performing an analytical processwhich includes a physical change in a substance, e.g., a sample,analyte, or reagent (sometimes referred to herein as “physicalanalysis”), performing an analytical method, e.g., a method whichincludes one or more of the following: separating or purifying asubstance, e.g., an analyte, or a fragment or other derivative thereof,from another substance; combining an analyte, or fragment or otherderivative thereof, with another substance, e.g., a buffer, solvent, orreactant; or changing the structure of an analyte, or a fragment orother derivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the analyte; orby changing the structure of a reagent, or a fragment or otherderivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the reagent.

“Acquiring a sample” as the term is used herein, refers to obtainingpossession of a sample, e.g., a tissue sample or nucleic acid sample, by“directly acquiring” or “indirectly acquiring” the sample. “Directlyacquiring a sample” means performing a process (e.g., performing aphysical method such as a surgery or extraction) to obtain the sample.“Indirectly acquiring a sample” refers to receiving the sample fromanother party or source (e.g., a third party laboratory that directlyacquired the sample). Directly acquiring a sample includes performing aprocess that includes a physical change in a physical substance, e.g., astarting material, such as a tissue, e.g., a tissue in a human patientor a tissue that has was previously isolated from a patient. Exemplarychanges include making a physical entity from a starting material,dissecting or scraping a tissue; separating or purifying a substance(e.g., a sample tissue or a nucleic acid sample); combining two or moreseparate entities into a mixture; performing a chemical reaction thatincludes breaking or forming a covalent or non-covalent bond. Directlyacquiring a sample includes performing a process that includes aphysical change in a sample or another substance, e.g., as describedabove.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific aspects, it is apparent that other aspects and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such aspects andequivalent variations.

Chromatography and Spectroscopy Techniques

The processing solutions can be used along with chromatographic and /orspectroscopic methods to further purify, or assess the purity ofsolutions. Methods of 1-dimensional (1D) chromatography suitable for usein the methods described here are known to one of skill in the art andinclude, e.g., affinity chromatography, gel filtration chromatography,ion exchange chromatography, reversed phase chromatography, hydrophobicinteraction chromatography. In some embodiments, the one-dimensionalchromatography method is HPLC reversed phase chromatography.Chromatography can include high performance liquid chromatography(HPLC), gas chromatography (GC), capillary electrophoresis, ionmobility. See also, e.g., Process Scale Purification of Antibodies, UweGottschalk 2011 John Wiley & Sons ISBN: 1118210743; Antibodies Vol 1Production and Purification, G. Subramanian 2013 Springer Science &Business Media; Basic Methods in Antibody Production andCharacterization, Gary C. Howard 2000 CRC Press.

Additional exemplary chromatographic methods include, but are notlimited to, Strong Anion Exchange chromatography (SAX), liquidchromatography (LC), high performance liquid chromatography (HPLC),ultra performance liquid chromatography (UPLC), thin layerchromatography (TLC), amide column chromatography, and combinationsthereof. Exemplary mass spectrometry (MS) include, but are not limitedto, tandem MS, LC-MS, LC-MS/MS, matrix assisted laser desorptionionisation mass spectrometry (MALDI-MS), Fourier transform massspectrometry (FTMS), ion mobility separation with mass spectrometry(IMS-MS), electron transfer dissociation (ETD-MS), and combinationsthereof. Exemplary electrophoretic methods include, but are not limitedto, capillary electrophoresis (CE), CE-MS, gel electrophoresis, agarosegel electrophoresis, acrylamide gel electrophoresis, SDS-polyacrylamidegel electrophoresis (SDS-PAGE) followed by Western blotting usingantibodies that recognize specific glycan structures, and combinationsthereof. Exemplary nuclear magnetic resonance (NMR) include, but are notlimited to, one-dimensional NMR (1D-NMR), two-dimensional NMR (2D-NMR),correlation spectroscopy magnetic-angle spinning NMR (COSY-NMR), totalcorrelated spectroscopy NMR (TOCSY-NMR), heteronuclear single-quantumcoherence NMR (HSQC-NMR), heteronuclear multiple quantum coherence(HMQC-NMR), rotational nuclear overhauser effect spectroscopy NMR(ROESY-NMR), nuclear overhauser effect spectroscopy (NOESY-NMR), andcombinations thereof.

Suitable spectroscopic techniques include, e.g., Raman spectroscopy.Raman spectroscopy is a technique that is increasing in popularity amongthe different disciplines of forensic science. Some examples of its usetoday involve the identification of drugs (Hodges et al., “The Use ofFourier Transform Raman Spectroscopy in the Forensic Identification ofIllicit Drugs and Explosives,” Molecular Spectroscopy 46:303-307(1990)), lipsticks (Rodger et al., “The In-Situ Analysis of Lipsticks bySurface Enhanced Resonance Raman Scattering,” Analyst 1823-1826 (1998)),and fibers (Thomas et al., “Raman Spectroscopy and the Forensic Analysisof Black/Grey and Blue Cotton Fibres Part 1: Investigation of theEffects of Varying Laser Wavelength,” Forensic Sci. Int. 152:189-197(2005)), as well as paint (Suzuki et al., “In Situ Identification andAnalysis of Automotive Paint Pigments Using Line Segment ExcitationRaman Spectroscopy: I. Inorganic Topcoat Pigments,” J. Forensic Sci.46:1053-1069 (2001)) and ink (Mazzella et al., “Raman Spectroscopy ofBlue Gel Pen Inks,” Forensic Sci. Int. 152:241-247 (2005)) analysis. Thetheory behind Raman spectroscopy is based on the inelastic scattering oflow-intensity, nondestructive laser light by a solid, liquid or gassample. Very little or no sample preparation is needed, and the requiredamount of tested material could be as low as several picograms orfemtoliters (10⁻¹² gram or 10⁻¹⁵ liter, respectively). A typical Ramanspectrum consists of several narrow bands and provides a uniquevibrational signature of the material (Grasselli et al., “ChemicalApplications of Raman Spectroscopy,” New York: John Wiley & Sons(1981)). Unlike infrared (IR) absorption spectroscopy, another type ofvibrational spectroscopy, Raman spectroscopy shows very littleinterference from water (Grasselli et al., “Chemical Applications ofRaman Spectroscopy,” New York: John Wiley & Sons (1981)), and that makesit a great technique for analyzing body fluids and their traces. ProperRaman spectroscopic measurements do not damage the sample. The stain orswab could be tested on the field and still be available for further usein the lab for DNA analysis, and that is very important to forensicapplication. The design of a portable Raman spectrometer is a realitynow (Yan et al., “Surface-Enhanced Raman Scattering Detection ofChemical and Biological Agents Using a Portable Raman Integrated TunableSensor,” Sensors and Actuators B. 6 (2007); Eckenrode et al., “PortableRaman Spectroscopy Systems for Field Analysis,” Forensic ScienceCommunications 3:(2001)) which would lead to the ability to makeidentifications at the crime scene.

The types of Raman spectroscopy suitable for use in conjunction with thepresent disclosure include, but are not limited to conventional Ramanspectroscopy, Raman microspectroscopy, near-field Raman spectroscopy,including but not limited to the tip-enhanced

Raman spectroscopy, surface enhanced Raman spectroscopy (SERS) andsurface enhanced resonance Raman spectroscopy (SERRS), coherentanti-Stokes Raman spectroscopy (CARS), etc. Both Stokes and anti-StokesRaman spectroscopy can be used.

In some embodiments, a rapid non-invasive test is performed on one ormore bioprocessing intermediates using the chromatographic orspectroscopic methods described herein. In some embodiments, the rapidtest is performed using Ramon spectroscopy.

Mass Spectrometry

Mass spectrometry methods suitable for use in the methods describedherein are known to one of skill in the art and include, e.g.,electrospray ionization MS, matrix-assisted laser desportion/ionizationMS, time of flight MS, fourier-transform ion cyclotron resonance MS,quadrupole time of flight MS, linear quadrupole, quadrupole ion trap MS,orbitrap, cylindrical ion trap, three dimensional ion trap, quadrupolemass filter, tandem mass spectrometry. In some embodiments, the massspectrometry is tandem mass spectrometry. See also, e.g., Protein MassSpectrometry, Julian Whitelegge 2008, Elsevier; Protein Sequencing andIdentification Using Tandem Mass Spectrometry, Michael Kinter 2005, JohnWiley & Sons; Characterization of Protein Therapeutics using MassSpectrometry, Guodong Chen 2014, Springer Science & Business Media.

Production Parameters

Methods described herein include determining and/or selecting aproduction parameter or parameters for a glycoprotein preparation suchthat a preselected glycan property or properties can be obtained uponproduction of a glycoprotein preparation. By using information regardingthe effects of various production parameters on glycosylation,production parameters can be selected prior to the production of aglycoprotein preparation that positively correlate with the desiredglycan properties. A production parameter as used herein is a parameteror element in a production process. Production parameters that can beselected include, e.g., the cell or cell line used to produce theglycoprotein preparation, the culture medium, culture process orbioreactor variables (e.g., batch, fed-batch, or perfusion),purification process and formulation of a glycoprotein preparation.

Primary production parameters include: 1) the types of host; 2) geneticsof the host; 3) media type; 4) fermentation platform; 5) purificationsteps; and 6) formulation. Secondary production parameter, as usedherein, is a production parameter that is adjustable or variable withineach of the primary production parameters. Examples include: selectionof host subclones based on desired glycan properties; regulation of hostgene levels constitutive or inducible; introduction of novel genes orpromoter elements; media additives (e.g. partial list on Table IV);physiochemical growth properties; growth vessel type (e.g. bioreactortype, T flask); cell density; cell cycle; enrichment of product with adesired glycan type (e.g. by lectin or antibody-mediated enrichment,ion-exchange chromatography, CE, or similar method); or similarsecondary production parameters clear to someone skilled in the art.

Media and Buffers

The methods described herein can include determining and/or selecting amedia component and/or the concentration of a media component that has apositive correlation to a desired glycan property or properties. A mediacomponent can be added in or administered over the course ofglycoprotein production or when there is a change media, depending onculture conditions. Media components include components added directlyto culture as well as components that are a byproduct of cell culture.

Media components include, e.g., buffer, amino acid content, vitamincontent, salt content, mineral content, serum content, carbon sourcecontent, lipid content, nucleic acid content, hormone content, traceelement content, ammonia content, co-factor content, indicator content,small molecule content, hydrolysate content and enzyme modulatorcontent.

Table 1 below provides examples of various media components that can beselected.

TABLE 1 Exemplary media components. amino acids sugar precursorsVitamins Indicators Carbon source (natural and Nucleosides ornucleotides unnatural) Salts butyrate or organics Sugars DMSO SeraAnimal derived products Plant derived hydrolysates Gene inducers sodiumpyruvate Non-natural sugars Surfactants Regulators of intracellular pHAmmonia Betaine or osmoprotectant Lipids Trace elements Hormones orgrowth factors minerals Buffers Non-natural amino acids Non-naturalamino acids Non-natural vitamins

Exemplary buffers include Tris, Tricine, HEPES, MOPS, PIPES, TAPS,bicine, BES, TES, cacodylate, MES, acetate, MKP, ADA, ACES, glycinamideand acetamidoglycine.

Minerals that are optionally present include bismuth, boron, calcium,chlorine, chromium, cobalt, copper, fluorine, iodine, iron, magnesium,manganese, molybdenum, nickel, phosphorus, potassium, rubidium,selenium, silicon, sodium, strontium, sulfur, tellurium, titanium,tungsten, vanadium, and zinc. Exemplary salts and minerals include CaCl2(anhydrous), CuSO4 5H2O, Fe(NO3).9H2O, KCl, KNO3, KH2PO4, MgSO4(anhydrous), NaCl, NaH2PO4H2O, NaHCO3, Na2SE3 (anhydrous), ZnSO4.7H2O;linoleic acid, lipoic acid, D-glucose, hypoxanthine 2Na, phenol red,putrescine 2HCl, sodium pyruvate, thymidine, pyruvic acid, sodiumsuccinate, succinic acid, succinic acid.Na.hexahydrate, glutathione(reduced), para-aminobenzoic acid (PABA), methyl linoleate, bactopeptone G, adenosine, cytidine, guanosine, 2′-deoxyadenosine HCl,2′-deoxycytidine HCl, 2′-deoxyguanosine and uridine. When the desiredglycan characteristic is decreased fucosylation, the productionparameters can include culturing a cell, e.g., CHO cell, e.g., dhfrdeficient CHO cell, in the presence of manganese, e.g., manganesepresent at a concentration of about 0.1 μM to 50 μM. Decreasedfucosylation can also be obtained, e.g., by culturing a cell (e.g., aCHO cell, e.g., a dhfr deficient CHO cell) at an osmolality of about 350to 500 mOsm. Osmolality can be adjusted by adding salt to the media orhaving salt be produced as a byproduct as evaporation occurs duringproduction.

Production parameters can also include physiochemical parameters. Suchconditions can include temperature, pH, osmolality, shear force oragitation rate, oxidation, spurge rate, growth vessel, tangential flow,DO, CO₂, nitrogen, fed batch, redox, cell density and feed strategy.Examples of physiochemical parameters that can be selected are providedin Table 2 below.

TABLE 2 Exemplary physiochemical parameters. pH CO₂ Osmolality Nitrogenshear force, or agitation rate Fed batch Oxidation Redox Spurge rateCell density Growth vessel Perfusion culture Tangential flow Feedstrategy Batch Temperature Dissolved O2 Time of culture

The system can be chosen based, at least in part, upon its correlationwith a desired glycan property or properties. Cells can be grown, forexample, as batch, fed-batch, perfusion, or continuous cultures.Production parameters that can be selected include, e.g., addition orremoval of media including when (early, middle or late during culturetime) and how often media is harvested; increasing or decreasing speedat which cell cultures are agitated; increasing or decreasingtemperature at which cells are cultured; adding or removing media suchthat culture density is adjusted; selecting a time at which cellcultures are started or stopped; and selecting a time at which cellculture parameters are changed. Such parameters can be selected for anyof the batch, fed-batch, perfusion and continuous culture conditions. Awide array of flasks, bottles, reactors, and controllers allow theproduction and scale up of cell culture systems.

Additional production parameters are known to one of skill in the art,see e.g., Antibody Expression and Production (2011) Ed. MohamedAl-Rubeai; Springer Publishing.

Products and Nucleic Acids Encoding Them

Provided herein are methods for identifying, selecting, or making a cellor cell line capable of producing a product. The products encompassed bythe present disclosure include, but are not limited to, molecules,nucleic acids, polypeptides (e.g., recombinant polypeptides, e.g.,antibodies, bispecific antibodies, multispecific antibodies), or hybridsthereof, that can be produced by, e.g., expressed in, a cell. In someembodiments, the cells are engineered or modified to produce theproduct. Such modifications include the introducing molecules thatcontrol or result in production of the product. For example, a cell ismodified by introducing an exogenous nucleic acid that encodes apolypeptide, e.g., a recombinant polypeptide, and the cell is culturedunder conditions suitable for production, e.g., expression andsecretion, of the polypeptide, e.g., recombinant polypeptide.

In embodiments, the cell or cell line identified, selected, or generatedby the methods described herein produces a product, e.g., a recombinantpolypeptide, useful in the treatment of a medical condition, disorder ordisease. Examples of medical conditions, disorders or diseases include,but are not limited to, metabolic disease or disorders (e.g., metabolicenzyme deficiencies), endocrine disorders (e.g., hormone deficiencies),haemostasis, thrombosis, hematopoietic disorders, pulmonary disorders,gastro-intestinal disorders, immunoregulation (e.g., immunodeficiency),infertility, transplantation, cancer, and infectious diseases.

In some embodiments, the product is an exogenous protein, e.g., aprotein that is not naturally expressed by the cell. The product can bea therapeutic protein or a diagnostic protein, e.g., useful for drugscreening. The therapeutic or diagnostic protein can be an antibodymolecule, e.g., an antibody or an antibody fragment, a fusion protein, ahormone, a cytokine, a growth factor, an enzyme, a glycoprotein, alipoprotein, a reporter protein, a therapeutic peptide, or a structuraland/or functional fragment or hybrid of any of these.

In one embodiment, the product, e.g., recombinant polypeptide, is anantibody molecule. Products encompassed herein comprise diagnostic andtherapeutic antibody molecules. A diagnostic antibody molecule includesan antibody, e.g., a monoclonal antibody or antibody fragment thereof,useful for imaging techniques. A therapeutic antibody molecule issuitable for administration to subjects, e.g., for treatment orprevention of a disease or disorder.

An antibody molecule is a protein, or polypeptide sequence derived froman immunoglobulin molecule which specifically binds with an antigen. Inan embodiment, the antibody molecule is a full-length antibody or anantibody fragment. Antibodies and multiformat proteins can be polyclonalor monoclonal, multiple or single chain, or intact immunoglobulins, andmay be derived from natural sources or from recombinant sources.Antibodies can be tetramers of immunoglobulin molecules. In anembodiment, the antibody is a monoclonal antibody. The antibody may be ahuman or humanized antibody. In one embodiment, the antibody is an IgA,IgG, IgD, or IgE antibody. In one embodiment, the antibody is an IgG1,IgG2, IgG3, or IgG4 antibody.

“Antibody fragment” refers to at least one portion of an intactantibody, or recombinant variants thereof, and refers to the antigenbinding domain, e.g., an antigenic determining variable region of anintact antibody, that is sufficient to confer recognition and specificbinding of the antibody fragment to a target, such as an antigen.Examples of antibody fragments include, but are not limited to, Fab,Fab′, F(ab′)₂, and Fv fragments, scFv antibody fragments, linearantibodies, single domain antibodies such as sdAb (either VL or VH),camelid VHH domains, and multi-specific antibodies formed from antibodyfragments such as a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region, and an isolated CDR orother epitope binding fragments of an antibody. An antigen bindingfragment can also be incorporated into single domain antibodies,maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies,tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, NatureBiotechnology 23:1126-1136, 2005). Antigen binding fragments can also begrafted into scaffolds based on polypeptides such as a fibronectin typeIII (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectinpolypeptide minibodies).

Exemplary products, e.g., polypeptides, e.g., recombinant polypeptides,produced in the methods or cells described herein are provided in Tables3 and 4 below.

TABLE 3 Therapeutic Product type Product Trade Name HormoneErythropoietin, Epoein-α Epogen, Procrit Darbepoetin-α Aranesp Growthhormone (GH), Genotropin, Humatrope, Norditropin, NovIVitropin,somatotropin Nutropin, Omnitrope, Protropin, Siazen, Serostim, ValtropinHuman follicle-stimulating Gonal-F, Follistim hormone (FSH) Humanchorionic gonadotropin Ovidrel Lutropin-α Luveris Glucagon GlcaGenGrowth hormone releasing Geref hormone (GHRH) Secretin ChiRhoStim (humanpeptide), SecreFlo (porcine peptide) Thyroid stimulating hormoneThyrogen (TSH), thyrotropin Blood Factor VIIa NovoSevenClotting/Coagulation Factor VIII Bioclate, Helixate, Kogenate,Recombinate, ReFacto Factors Factor IX Benefix Antithrombin III (AT-III)Thrombate III Protein C concentrate Ceprotin Cytokine/Growth Type Ialpha-interferon Infergen factor Interferon-αn3 (IFNαn3) Alferon NInterferon-β1a (rIFN-β) Avonex, Rebif Interferon-β1b (rIFN-β) BetaseronInterferon-γ1b (IFN γ) Actimmune Aldesleukin (interleukin 2(IL2),Proleukin epidermal theymocyte activating factor; ETAF Palifermin(keratinocyte growth Kepivance factor; KGF) Becaplemin (platelet-derivedRegranex growth factor; PDGF) Anakinra (recombinant IL1 Anril, Kineretantagonist) Antibody molecules Bevacizumab (VEGFA mAb) Avastin Cetuximab(EGFR mAb) Erbitux Panitumumab (EGFR mAb) Vectibix Alemtuzumab (CD52mAb) Campath Rituximab (CD20 chimeric Ab) Rituxan Trastuzumab (HER2/NeumAb) Herceptin Abatacept (CTLA Ab/Fc fusion) Orencia Adalimumab (TNFαmAb) Humira Etanercept (TNF receptor/Fc Enbrel fusion) Infliximab (TNFαchimeric mAb) Remicade Alefacept (CD2 fusion protein) Amevive Efalizumab(CD11a mAb) Raptiva Natalizumab (integrin α4 subunit Tysabri mAb)Eculizumab (C5mAb) Soliris Muromonab-CD3 Orthoclone, OKT3 Other: InsulinHumulin, Novolin Fusion Hepatitis B surface antigen Engerix, RecombivaxHB proteins/Protein (HBsAg) vaccines/Peptides HPV vaccine Gardasil OspALYMErix Anti-Rhesus(Rh) immunoglobulin G Rhophylac Enfuvirtide FuzeonSpider silk, e.g., fibrion QMONOS

In another embodiment, the product is a bispecific molecule, e.g., abispecific antibody. Bispecific molecules, as described herein, includemolecules that can bind to two or more distinct antigens or targets. Inan embodiment, a bispecific molecule comprises antibody fragments. Inone embodiment, the bispecific molecule comprises a bispecific antibody,a bispecific antibody fusion protein, or a bispecific antibodyconjugate, a Bi-specific T cell Engager (BiTE) molecule, a Dual AffinityRe-Targeting (DART) Molecule, a Dual Action Fab (DAF) molecule, ananobody, or other arrangement of antibody fragments resulting in amolecule having the ability to recognize or bind to two distinctantigens.

TABLE 4 Exemplary Products, e.g., Bispecific Molecules BsAb (othernames, sponsoring Proposed mechanisms Development Diseases (ororganizations) BsAb format Targets of action stages healthy volunteers)Catumaxomab BsIgG: CD3, Retargeting of T cells to Approved in Malignantascites in (Removab ®, Fresenius Triomab EpCAM tumor, Fc mediated EUEpCAM positive Biotech, Trion Pharma, effector functions tumorsNeopharm) Ertumaxomab (Neovii BsIgG: CD3, HER2 Retargeting of T cells toPhase I/II Advanced solid Biotech, Fresenius Triomab tumor tumorsBiotech) Blinatumomab BiTE CD3, CD19 Retargeting of T cells to Approvedin Precursor B-cell (Blincyto ®, AMG 103, tumor USA ALL MT 103, MEDI538, Phase II and ALL Amgen) III DLBCL Phase II NHL Phase I REGN1979(Regeneron) BsAb CD3, CD20 Solitomab (AMG 110, BiTE CD3, Retargeting ofT cells to Phase I Solid tumors MT110, Amgen) EpCAM tumor MEDI 565 (AMG211, BiTE CD3, CEA Retargeting of T cells to Phase I GastrointestinalMedImmune, Amgen) tumor adenocancinoma RO6958688 (Roche) BsAb CD3, CEABAY2010112 (AMG BiTE CD3, PSMA Retargeting of T cells to Phase IProstate cancer 212, Bayer; Amgen) tumor MGD006 (Macrogenics) DART CD3,CD123 Retargeting of T cells to Phase I AML tumor MGD007 (Macrogenics)DART CD3, gpA33 Retargeting of T cells to Phase I Colorectal cancertumor MGD011 (Macrogenics) DART CD19, CD3 SCORPION (Emergent BsAb CD3,CD19 Retargeting of T cells to Biosolutions, Trubion) tumor AFM11(Affimed TandAb CD3, CD19 Retargeting of T cells to Phase I NHL and ALLTherapeutics) tumor AFM12 (Affimed TandAb CD19, CD16 Retargeting of NKcells Therapeutics) to tumor cells AFM13 (Affimed TandAb CD30,Retargeting of NK cells Phase II Hodgkin's Therapeutics) CD16A to tumorcells Lymphoma GD2 (Barbara Ann T cells CD3, GD2 Retargeting of T cellsto Phase I/II Neuroblastoma and Karmanos Cancer preloaded tumorosteosarcoma Institute) with BsAb pGD2 (Barbara Ann T cells CD3, Her2Retargeting of T cells to Phase II Metastatic breast Karmanos Cancerpreloaded tumor cancer Institute) with BsAb EGFRBi-armed T cells CD3,EGFR Autologous activated T Phase I Lung and other autologous activatedT preloaded cells to EGFR-positive solid tumors cells (Roger Williamswith BsAb tumor Medical Center) Anti-EGFR-armed T cells CD3, EGFRAutologous activated T Phase I Colon and activated T-cells preloadedcells to EGFR-positive pancreatic cancers (Barbara Ann Karmanos withBsAb tumor Cancer Institute) rM28 (University Tandem scFv CD28,Retargeting of T cells to Phase II Metastatic Hospital Tubingen) MAPGtumor melanoma IMCgp100 ImmTAC CD3, peptide Retargeting of T cells toPhase I/II Metastatic (Immunocore) MHC tumor melanoma DT2219ARL (NCI, 2scFv linked CD19, CD22 Targeting of protein Phase I B cell leukemiaUniversity of Minnesota) to diphtheria toxin to tumor or lymphoma toxinXmAb5871 (Xencor) BsAb CD19, CD32b NI-1701 (NovImmune) BsAb CD47, CD19MM-111 (Merrimack) BsAb ErbB2, ErbB3 MM-141 (Merrimack) BsAb IGF-1R,ErbB3 NA (Merus) BsAb HER2, HER3 NA (Merus) BsAb CD3, CLEC12A NA (Merus)BsAb EGFR, HER3 NA (Merus) BsAb PD1, undisclosed NA (Merus) BsAb CD3,undisclosed Duligotuzumab DAF EGFR, HER3 Blockade of 2 receptors, PhaseI and II Head and neck (MEHD7945A, ADCC Phase II cancer Genentech,Roche) Colorectal cancer LY3164530 (Eli Lily) Not disclosed EGFR, METBlockade of 2 receptors Phase I Advanced or metastatic cancer MM-111(Merrimack HSA body HER2, HER3 Blockade of 2 receptors Phase II Gastricand Pharmaceuticals) Phase I esophageal cancers Breast cancer MM-141,(Merrimack IgG-scFv IGF-1R, Blockade of 2 receptors Phase I Advancedsolid Pharmaceuticals) HER3 tumors RG7221 (RO5520985, CrossMab Ang2,VEGF A Blockade of 2 Phase I Solid tumors Roche) proangiogenics RG7716(Roche) CrossMab Ang2, VEGF A Blockade of 2 Phase I Wet AMDproangiogenics OMP-305B83 BsAb DLL4/VEGF (OncoMed) TF2 (Immunomedics)Dock and CEA, HSG Pretargeting tumor for Phase II Colorectal, breastlock PET or radioimaging and lung cancers ABT-981 (AbbVie) DVD-Ig IL-1α,IL-1β Blockade of 2 Phase II Osteoarthritis proinflammatory cytokinesABT-122 (AbbVie) DVD-Ig TNF, IL-17A Blockade of 2 Phase II Rheumatoidproinflammatory arthritis cytokines COVA322 IgG-fynomer TNF, IL17ABlockade of 2 Phase I/II Plaque psoriasis proinflammatory cytokinesSAR156597 (Sanofi) Tetravalent IL-13, IL-4 Blockade of 2 Phase IIdiopathic bispecific proinflammatory pulmonary fibrosis tandem IgGcytokines GSK2434735 Dual- IL-13, IL-4 Blockade of 2 Phase I (Healthy(GSK) targeting proinflammatory volunteers) domain cytokinesOzoralizumab (ATN103, Nanobody TNF, HSA Blockade of Phase II RheumatoidAblynx) proinflammatory arthritis cytokine, binds to HSA to increasehalf-life ALX-0761 (Merck Nanobody IL-17A/F, Blockade of 2 Phase I(Healthy Serono, Ablynx) HSA proinflammatory volunteers) cytokines,binds to HSA to increase half-life ALX-0061 (AbbVie, Nanobody IL-6R, HSABlockade of Phase I/II Rheumatoid Ablynx; proinflammatory arthritiscytokine, binds to HSA to increase half-life ALX-0141 (Ablynx, NanobodyRANKL, Blockade of bone Phase I Postmenopausal Eddingpharm) HSAresorption, binds to bone loss HSA to increase half- life RG6013/ACE910ART-Ig Factor IXa, Plasma coagulation Phase II Hemophilia (Chugai,Roche) factor X

Other exemplary therapeutic or diagnostic proteins include, but are notlimited to any protein described in Tables 1-10 of Leader et al.,“Protein therapeutics: a summary and pharmacological classification”,Nature Reviews Drug Discovery, 2008, 7:21-39 (incorporated herein byreference); or any conjugate, variant, analog, or functional fragment ofthe recombinant polypeptides described herein.

Other recombinant products include non-antibody scaffolds or alternativeprotein scaffolds, such as, but not limited to: DARPins, affibodies andadnectins. Such non-antibody scaffolds or alternative protein scaffoldscan be engineered to recognize or bind to one or two, or more, e.g., 1,2, 3, 4, or 5 or more, different targets or antigens. Also providedherein are nucleic acids, e.g., exogenous nucleic acids that encode theproducts, e.g., polypeptides, e.g., recombinant polypeptides describedherein. The nucleic acid sequences coding for the desired recombinantpolypeptides can be obtained using recombinant methods known in the art,such as, for example by screening libraries from cells expressing thedesired nucleic acid sequence, e.g., gene, by deriving the nucleic acidsequence from a vector known to include the same, or by isolatingdirectly from cells and tissues containing the same, using standardtechniques. Alternatively, the nucleic acid encoding the recombinantpolypeptide can be produced synthetically, rather than cloned.Recombinant DNA techniques and technology are highly advanced and wellestablished in the art. Accordingly, the ordinarily skilled artisanhaving the knowledge of the amino acid sequence of a recombinantpolypeptide described herein can readily envision or generate thenucleic acid sequence that would encode the recombinant polypeptide.

In some embodiments, the exogenous nucleic acid controls the expressionof a product that is endogenously expressed by the host cell. In suchembodiments, the exogenous nucleic acid comprises one or more nucleicacid sequences that increase the expression of the endogenous product(also referred to herein as “endogenous product transactivationsequence”). For example, the nucleic acid sequence that increases theexpression of an endogenous product comprises a constitutively activepromoter or a promoter that is stronger, e.g., increases transcriptionat the desired site, e.g., increases expression of the desiredendogenous gene product. After introduction of the exogenous nucleicacid comprising the endogenous product transactivation sequence, saidexogenous nucleic acid is integrated into the chromosomal genome of thecell, e.g., at a preselected location proximal to the genomic sequenceencoding the endogenous product, such that the endogenous producttransactivation sequence increases the transactivation or expression ofthe desired endogenous product. Other methods for modifying a cell,e.g., introducing an exogenous nucleic acid, for increasing expressionof an endogenous product is described, e.g., in U.S. Pat. No. 5,272,071;hereby incorporated by reference in its entirety.

The expression of a product described herein is typically achieved byoperably linking a nucleic acid encoding the recombinant polypeptide orportions thereof to a promoter, and incorporating the construct into anexpression vector. The vectors can be suitable for replication andintegration eukaryotes or prokaryotes. Typical cloning vectors containother regulatory elements, such as transcription and translationterminators, initiation sequences, and promoters useful for regulationof the expression of the desired nucleic acid sequence.

The nucleic acid sequences described herein encoding a product, e.g., arecombinant polypeptide, or comprising a nucleic acid sequence that cancontrol the expression of an endogenous product, can be cloned into anumber of types of vectors. For example, the nucleic acid can be clonedinto a vector including, but not limited to a plasmid, a phagemid, aphage derivative, an animal virus, and a cosmid. Vectors of particularinterest include expression vectors, replication vectors, probegeneration vectors, and sequencing vectors. In embodiments, theexpression vector may be provided to a cell in the form of a viralvector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al., 2012, MOLECULAR CLONING: ALABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193). Vectors derivedfrom viruses are suitable tools to achieve long-term gene transfer sincethey allow long-term, stable integration of a transgene and itspropagation in daughter cells.

A vector may also include, e.g., a signal sequence to facilitatesecretion, a polyadenylation signal and transcription terminator (e.g.,from Bovine Growth Hormone (BGH) gene), an element allowing episomalreplication and replication in prokaryotes (e.g. SV40 origin and ColE1or others known in the art) and/or elements to allow selection, e.g., aselection marker or a reporter gene.

In one embodiment, the vector comprising a nucleic acid sequenceencoding a polypeptide, e.g., a recombinant polypeptide, furthercomprises a promoter sequence responsible for the recruitment ofpolymerase to enable transcription initiation for expression of thepolypeptide, e.g., the recombinant polypeptide. In one embodiment,promoter sequences suitable for the methods described herein are usuallyassociated with enhancers to drive high amounts of transcription andhence deliver large copies of the target exogenous mRNA. In anembodiment, the promoter comprises cytomegalovirus (CMV) major immediateearly promoters (Xia, Bringmann et al. 2006) and the SV40 promoter(Chernajovsky, Mory et al. 1984), both derived from their namesakeviruses or promoters derived therefrom. Several other less common viralpromoters have been successfully employed to drive transcription uponinclusion in an expression vector including Rous Sarcoma virus longterminal repeat (RSV-LTR) and Moloney murine leukaemia virus (MoMLV) LTR(Papadakis, Nicklin et al. 2004). In another embodiment, specificendogenous mammalian promoters can be utilized to drive constitutivetranscription of a gene of interest (Pontiller, Gross et al. 2008). TheCHO specific Chinese Hamster elongation factor 1-alpha (CHEF1α) promoterhas provided a high yielding alternative to viral based sequences (Deer,Allison 2004). In addition to promoters, the vectors described hereinfurther comprise an enhancer region as described above; a specificnucleotide motif region, proximal to the core promoter, which canrecruit transcription factors to upregulate the rate of transcription(Riethoven 2010). Similar to promoter sequences, these regions are oftenderived from viruses and are encompassed within the promoter sequencesuch as hCMV and SV40 enhancer sequences, or may be additionallyincluded such as adenovirus derived sequences (Gaillet, Gilbert et al.2007).

In one embodiment, the vector comprising a nucleic acid sequenceencoding a product, e.g., a polypeptide, e.g., a recombinantpolypeptide, described herein further comprises a nucleic acid sequencethat encodes a selection marker. In one embodiment, the selectablemarker comprises glutamine synthetase (GS); dihydrofolate reductase(DHFR) e.g., an enzyme which confers resistance to methotrexate (MTX);or an antibiotic marker, e.g., an enzyme that confers resistance to anantibiotic such as: hygromycin, neomycin (G418), zeocin, puromycin, orblasticidin. In another embodiment, the selection marker comprises or iscompatible with the Selexis selection system (e.g., SUREtechnologyPlatform™ and Selexis Genetic Elements™ commercially available fromSelexis SA) or the Catalant selection system.

In one embodiment, the vector comprising a nucleic acid sequenceencoding a recombinant product described herein comprises a selectionmarker that is useful in identifying a cell or cells comprise thenucleic acid encoding a recombinant product described herein. In anotherembodiment, the selection marker is useful in identifying a cell orcells that comprise the integration of the nucleic acid sequenceencoding the recombinant product into the genome, as described herein.The identification of a cell or cells that have integrated the nucleicacid sequence encoding the recombinant protein can be useful for theselection and engineering of a cell or cell line that stably expressesthe product.

Suitable vectors for use are commercially available, and include vectorsassociated with the GS Expression System™, GS Xceed™ Gene ExpressionSystem, or Potelligent® CHOK1SV technology available from LonzaBiologics, Inc, e.g., vectors as described in Fan et al., Pharm.Bioprocess. (2013); 1(5):487-502, which is incorporated herein byreference in its entirety. GS expression vectors comprise the GS gene,or a functional fragment thereof (e.g., a GS mini-gene), and one ormore, e.g., 1, 2, or 3, or more, highly efficient transcriptioncassettes for expression of the gene of interest, e.g., a nucleic acidencoding a recombinant polypeptide described herein. A GS mini-genecomprises, e.g., consists of, intron 6 of the genomic CHO GS gene. Inone embodiment, a GS vector comprises a GS gene operably linked to aSV4OL promoter and one or two polyA signals. In another embodiment, a GSvector comprises a GS gene operably linked to a SV40E promoter, SV40splicing and polyadenylation signals. In such embodiments, thetranscription cassette, e.g., for expression of the gene of interest orrecombinant polypeptide described herein, includes the hCMV-MIE promoterand 5′ untranslated sequences from the hCMV-MIE gene including the firstintron. Other vectors can be constructed based on GS expression vectors,e.g., wherein other selection markers are substituted for the GS gene inthe expression vectors described herein.

Vectors suitable for use in the methods described herein include, butare not limited to, other commercially available vectors, such as,pcDNA3.1/Zeo, pcDNA3.1/CAT, pcDNA3.3TOPO (Thermo Fisher, previouslyInvitrogen); pTarget, HaloTag (Promega); pUC57 (GenScript); pFLAG-CMV(Sigma-Aldrich); pCMV6 (Origene); pEE12 or pEE14 (Lonza Biologics), orpBK-CMV/pCMV-3Tag-7/pCMV-Tag2B (Stratagene).

Cells and Cell Culture

In embodiments, the cell is a mammalian cell. In other embodiments, thecell is a cell other than a mammalian cell. In an embodiment, the cellis a mouse, rat, Chinese hamster, Syrian hamster, monkey, ape, dog,horse, ferret, or cat. In embodiments, the cell is a mammalian cell,e.g., a human cell or a rodent cell, e.g., a hamster cell, a mouse cell,or a rat cell. In another embodiment, the cell is from a duck, parrot,fish, insect, plant, fungus, or yeast. In one embodiment, the cell is anArchaebacteria. In an embodiment, the cell is a species ofActinobacteria, e.g., Mycobacterium tuberculosis).

In one embodiment, the cell is a Chinese hamster ovary (CHO) cell. Inone embodiment, the cell is a CHO-K1 cell, a CHO-K1 SV cell, a DG44 CHOcell, a DUXB11 CHO cell, a CHOS, a CHO GS knock-out cell, a CHO FUT8 GSknock-out cell, a CHOZN, or a CHO-derived cell. The CHO GS knock-outcell (e.g., GSKO cell) is, for example, a CHO-K1SV GS knockout cell(Lonza Biologics, Inc.). The CHO FUT8 knockout cell is, for example, thePotelligent® CHOK1 SV (Lonza Biologics, Inc.).

In another embodiment, the cell is a Hela, HEK293, HT1080, H9, HepG2,MCF7, Jurkat, NIH3T3, PC12, PER.C6, BHK (baby hamster kidney cell),VERO, SP2/0, NS0, YB2/0, Y0, EB66, C127, L cell, COS, e.g., COS1 andCOST, QC1-3, CHOK1, CHOK1SV, Potelligent CHOK1SV, CHO GS knockout,CHOK1SV GS-KO, CHOS, CHO DG44, CHO DXB11, and CHOZN, or any cellsderived therefrom. In one embodiment, the cell is a stem cell. In oneembodiment, the cell is a differentiated form of any of the cellsdescribed herein. In one embodiment, the cell is a cell derived from anyprimary cell in culture.

In an embodiment, the cell is any one of the cells described herein thatcomprises an exogenous nucleic acid encoding a recombinant polypeptide,e.g., expresses a recombinant polypeptide, e.g., a recombinantpolypeptide selected from Table 5 or 3.

In an embodiment, the cell culture is carried out as a batch culture,fed-batch culture, draw and fill culture, or a continuous culture. In anembodiment, the cell culture is a suspension culture. In one embodiment,the cell or cell culture is placed in vivo for expression of therecombinant polypeptide, e.g., placed in a model organism or a humansubject. In one embodiment, the culture media is free of serum.Serum-free and protein-free media are commercially available, e.g.,Lonza Biologics.

Suitable media and culture methods for mammalian cell lines arewell-known in the art, as described in U.S. Pat. No. 5,633,162, forinstance. Examples of standard cell culture media for laboratory flaskor low density cell culture and being adapted to the needs of particularcell types are for instance: Roswell Park Memorial Institute (RPMI) 1640medium (Morre, G., The Journal of the American Medical Association, 199,p. 519 f. 1967), L-15 medium (Leibovitz, A. et al., Amer. J. of Hygiene,78, 1p. 173 ff, 1963), Dulbecco's modified Eagle's medium (DMEM),Eagle's minimal essential medium (MEM), Ham's F12 medium (Ham, R. etal., Proc. Natl. Acad. Sc.53, p 288 ff. 1965) or Iscoves' modified DMEMlacking albumin, transferrin and lecithin (Iscoves et al., J. Exp. med.1, p. 923 ff., 1978). For instance, Ham's F10 or F12 media werespecially designed for CHO cell culture. Other media specially adaptedto CHO cell culture are described in EP-481 791. It is known that suchculture media can be supplemented with fetal bovine serum (FBS, alsocalled fetal calf serum FCS), the latter providing a natural source of aplethora of hormones and growth factors. The cell culture of mammaliancells is nowadays a routine operation well-described in scientifictextbooks and manuals, it is covered in detail e.g. in R. Ian Fresney,Culture of Animal cells, a manual, 4^(th) edition, Wiley-Liss/N.Y.,2000.

Other suitable cultivation methods are known to the skilled artisan andmay depend upon the recombinant polypeptide product and the host cellutilized. It is within the skill of an ordinarily skilled artisan todetermine or optimize conditions suitable for the expression andproduction of the recombinant polypeptide to be expressed by the cell.

In one aspect, the cell or cell line comprises an exogenous nucleic acidthat encodes a product, e.g., a recombinant polypeptide. In anembodiment, the cell or cell line expresses the product, e.g., atherapeutic or diagnostic product. Methods for genetically modifying orengineering a cell to express a desired polypeptide or protein are wellknown in the art, and include, for example, transfection, transduction(e.g., viral transduction), or electroporation.

Physical methods for introducing a nucleic acid, e.g., an exogenousnucleic acid or vector described herein, into a host cell includecalcium phosphate precipitation, lipofection, particle bombardment,microinjection, electroporation, and the like. Methods for producingcells comprising vectors and/or exogenous nucleic acids are well-knownin the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING:A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY).

Chemical means for introducing a nucleic acid, e.g., an exogenousnucleic acid or vector described herein, into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle). Other methodsof state-of-the-art targeted delivery of nucleic acids are available,such as delivery of polynucleotides with targeted nanoparticles or othersuitable sub-micron sized delivery system.

In embodiments, the integration of the exogenous nucleic acid into anucleic acid of the host cell, e.g., the genome or chromosomal nucleicacid of the host cell is desired. Methods for determining whetherintegration of an exogenous nucleic acid into the genome of the hostcell has occurred can include a GS/MSX selection method. The GS/MSXselection method uses complementation of a glutamine auxotrophy by arecombinant GS gene to select for high-level expression of proteins fromcells. Briefly, the GS/MSX selection method comprises inclusion of anucleic acid encoding glutamine synthetase on the vector comprising theexogenous nucleic acid encoding the recombinant polypeptide product.Administration of methionine sulfoximine (MSX) selects cells that havestably integrated into the genome the exogenous nucleic acid encodingboth the recombinant polypeptide and GS. As GS can be endogenouslyexpressed by some host cells, e.g., CHO cells, the concentration andduration of selection with MSX can be optimized to identify highproducing cells with stable integration of the exogenous nucleic acidencoding the recombinant polypeptide product into the host genome. TheGS selection and systems thereof is further described in Fan et al.,Pharm. Bioprocess. (2013); 1(5):487-502, which is incorporated herein byreference in its entirety.

Other methods for identifying and selecting cells that have stablyintegrated the exogenous nucleic acid into the host cell genome caninclude, but are not limited to, inclusion of a reporter gene on theexogenous nucleic acid and assessment of the presence of the reportergene in the cell, and PCR analysis and detection of the exogenousnucleic acid. In one embodiment, the cells selected, identified, orgenerated using the methods described herein are capable of producinghigher yields of protein product than cells that are selected using onlya selection method for the stable expression, e.g., integration ofexogenous nucleic acid encoding the recombinant polypeptide. In anembodiment, the cells selected, identified, or generated using themethods described herein produce 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, or 10-fold or more of the product, e.g.,recombinant polypeptide, as compared to cells that were not contactedwith an inhibitor of protein degradation, or cells that were onlyselected for stable expression, e.g., integration, of the exogenousnucleic acid encoding the recombinant polypeptide.

Methods for Cell Line and Recombinant Polypeptide Production

The current state of the art in both mammalian and microbial selectionsystems is to apply selective pressure at the level of the transcriptionof DNA into RNA. The gene of interest is tightly linked to the selectionmarker making a high level of expression of the selective marker likelyto result in the high expression of the gene of interest. Cells whichexpress the selection marker at high levels are able to survive andproliferate, those which do not are less likely to survive andproliferate, e.g., apoptose and/or die. In this way a population ofcells can be enriched for cells expressing the selection marker and byimplication the gene of interest at high levels. This method has provedvery successful for expressing straightforward proteins. In embodiments,the process described herein provides a substantially pure proteinproduct. As used herein, “substantially pure” is meant substantiallyfree of pyrogenic materials, substantially free of nucleic acids, and/orsubstantially free of endogenous cellular proteins enzymes andcomponents from the host cell, such as polymerases, ribosomal proteins,and chaperone proteins. A substantially pure protein product contains,for example, less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, or 1% of contaminating endogenous protein, nucleic acid, or othermacromolecule from the host cell.

The methods and systems can be used with methods for recovering andpurifying products that are well established in the art. For recoveringthe recombinant polypeptide product, a physical or chemical orphysical-chemical method is used. The physical or chemical orphysical-chemical method can be a filtering method, a centrifugationmethod, an ultracentrifugation method, an extraction method, alyophilization method, a precipitation method, a crystallization method,a chromatography method or a combination of two or more methods thereof.In an embodiment, the chromatography method comprises one or more ofsize-exclusion chromatography (or gel filtration), ion exchangechromatography, e.g., anion or cation exchange chromatography, affinitychromatography, hydrophobic interaction chromatography, and/ormultimodal chromatography.

The devices, facilities and methods described herein are suitable forculturing any desired cell line including prokaryotic and/or eukaryoticcell lines. Further, in embodiments, the devices, facilities and methodsare suitable for culturing suspension cells or anchorage-dependent(adherent) cells and are suitable for production operations configuredfor production of pharmaceutical and biopharmaceutical products—such aspolypeptide products, nucleic acid products (for example DNA or RNA), orcells and/or viruses such as those used in cellular and/or viraltherapies In embodiments, the cells express or produce a product, suchas a recombinant therapeutic or diagnostic product. As described in moredetail below, examples of products produced by cells include, but arenot limited to, antibody molecules (e.g., monoclonal antibodies,bispecific antibodies), antibody mimetics (polypeptide molecules thatbind specifically to antigens but that are not structurally related toantibodies such as e.g. DARPins, affibodies, adnectins, or IgNARs),fusion proteins (e.g., Fc fusion proteins, chimeric cytokines), otherrecombinant proteins (e.g., glycosylated proteins, enzymes, hormones),viral therapeutics (e.g., anti-cancer oncolytic viruses, viral vectorsfor gene therapy and viral immunotherapy), cell therapeutics (e.g.,pluripotent stem cells, mesenchymal stem cells and adult stem cells),vaccines or lipid-encapsulated particles (e.g., exosomes, virus-likeparticles), RNA (such as e.g. siRNA) or DNA (such as e.g. plasmid DNA),antibiotics or amino acids. In embodiments, the devices, facilities andmethods can be used for producing biosimilars.

As mentioned, in embodiments, devices, facilities and methods allow forthe production of eukaryotic cells, e.g., mammalian cells or lowereukaryotic cells such as for example yeast cells or filamentous fungicells, or prokaryotic cells such as Gram-positive or Gram-negative cellsand/or products of the eukaryotic or prokaryotic cells, e.g., proteins,peptides, antibiotics, amino acids, nucleic acids (such as DNA or RNA),synthesised by the eukaryotic cells in a large-scale manner. Unlessstated otherwise herein, the devices, facilities, and methods caninclude any desired volume or production capacity including but notlimited to bench-scale, pilot-scale, and full production scalecapacities.

Moreover and unless stated otherwise herein, the devices, facilities,and methods can include any suitable reactor(s) including but notlimited to stirred tank, airlift, fiber, microfiber, hollow fiber,ceramic matrix, fluidized bed, fixed bed, and/or spouted bedbioreactors. As used herein, “reactor” can include a fermentor orfermentation unit, or any other reaction vessel and the term “reactor”is used interchangeably with “fermentor.” For example, in some aspects,an example bioreactor unit can perform one or more, or all, of thefollowing: feeding of nutrients and/or carbon sources, injection ofsuitable gas (e.g., oxygen), inlet and outlet flow of fermentation orcell culture medium, separation of gas and liquid phases, maintenance oftemperature, maintenance of oxygen and CO2 levels, maintenance of pHlevel, agitation (e.g., stirring), and/or cleaning/sterilizing. Examplereactor units, such as a fermentation unit, may contain multiplereactors within the unit, for example the unit can have 1, 2, 3, 4, 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100, or morebioreactors in each unit and/or a facility may contain multiple unitshaving a single or multiple reacotrs within the facility. In variousembodiments, the bioreactor can be suitable for batch, semi fed-batch,fed-batch, perfusion, and/or a continuous fermentation processes. Anysuitable reactor diameter can be used. In embodiments, the bioreactorcan have a volume between about 100 mL and about 50,000 L. Non-limitingexamples include a volume of 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400liters, 450 liters, 500 liters, 550 liters, 600 liters, 650 liters, 700liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters,4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and/or50,000 liters. Additionally, suitable reactors can be multi-use,single-use, disposable, or non-disposable and can be formed of anysuitable material including metal alloys such as stainless steel (e.g.,316L or any other suitable stainless steel) and Inconel, plastics,and/or glass.

In embodiments and unless stated otherwise herein, the devices,facilities, and methods described herein can also include any suitableunit operation and/or equipment not otherwise mentioned, such asoperations and/or equipment for separation, purification, and isolationof such products. Any suitable facility and environment can be used,such as traditional stick-built facilities, modular, mobile andtemporary facilities, or any other suitable construction, facility,and/or layout. For example, in some embodiments modular clean-rooms canbe used. Additionally and unless otherwise stated, the devices, systems,and methods described herein can be housed and/or performed in a singlelocation or facility or alternatively be housed and/or performed atseparate or multiple locations and/or facilities.

By way of non-limiting examples and without limitation, U.S. PublicationNos. 2013/0280797; 2012/0077429; 2009/0305626; and U.S. Pat. Nos.8,298,054; 7,629,167; and 5,656,491, which are hereby incorporated byreference in their entirety, describe example facilities, equipment,and/or systems that may be suitable.

In embodiments, the cells are eukaryotic cells, e.g., mammalian cells.The mammalian cells can be for example human or rodent or bovine celllines or cell strains. Examples of such cells, cell lines or cellstrains are e.g. mouse myeloma (NSO)-cell lines, Chinese hamster ovary(CHO)-cell lines, HT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12,BHK (baby hamster kidney cell), VERO, SP2/0, YB2/0, Y0, C127, L cell,COS, e.g., COS 1 and COS7, QC1-3,HEK-293, VERO, PER.C6, HeLA, EB1, EB2,EB3, oncolytic or hybridoma-cell lines. Preferably the mammalian cellsare CHO-cell lines. In one embodiment, the cell is a CHO cell. In oneembodiment, the cell is a CHO-K1 cell, a CHO-K1 SV cell, a DG44 CHOcell, a DUXB11 CHO cell, a CHOS, a CHO GS knock-out cell, a CHO FUT8 GSknock-out cell, a CHOZN, or a CHO-derived cell. The CHO GS knock-outcell (e.g., GSKO cell) is, for example, a CHO-K1 SV GS knockout cell.The CHO FUT8 knockout cell is, for example, the Potelligent® CHOK1 SV(Lonza Biologics, Inc.). Eukaryotic cells can also be avian cells, celllines or cell strains, such as for example, EBx® cells, EB14, EB24,EB26, EB66, or EBv13.

In one embodiment, the eukaryotic cells are stem cells. The stem cellscan be, for example, pluripotent stem cells, including embryonic stemcells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs),tissue specific stem cells (e.g., hematopoietic stem cells) andmesenchymal stem cells (MSCs).

In one embodiment, the cell is a differentiated form of any of the cellsdescribed herein. In one embodiment, the cell is a cell derived from anyprimary cell in culture.

In embodiments, the cell is a hepatocyte such as a human hepatocyte,animal hepatocyte, or a non-parenchymal cell. For example, the cell canbe a plateable metabolism qualified human hepatocyte, a plateableinduction qualified human hepatocyte, plateable Qualyst TransporterCertified™ human hepatocyte, suspension qualified human hepatocyte(including 10-donor and 20-donor pooled hepatocytes), human hepatickupffer cells, human hepatic stellate cells, dog hepatocytes (includingsingle and pooled Beagle hepatocytes), mouse hepatocytes (including CD-1and C57BI/6 hepatocytes), rat hepatocytes (including Sprague-Dawley,Wistar Han, and Wistar hepatocytes), monkey hepatocytes (includingCynomolgus or Rhesus monkey hepatocytes), cat hepatocytes (includingDomestic Shorthair hepatocytes), and rabbit hepatocytes (including NewZealand White hepatocytes). Example hepatocytes are commerciallyavailable from Triangle Research Labs, LLC, 6 Davis Drive ResearchTriangle Park, N.C., USA 27709.

In one embodiment, the eukaryotic cell is a lower eukaryotic cell suchas e.g. a yeast cell (e.g., Pichia genus (e.g. Pichia pastoris, Pichiamethanolica, Pichia kluyveri, and Pichia angusta), Komagataella genus(e.g. Komagataella pastoris, Komagataella pseudopastoris or Komagataellaphaffii), Saccharomyces genus (e.g. Saccharomyces cerevisae, cerevisiae,Saccharomyces kluyveri, Saccharomyces uvarum), Kluyveromyces genus (e.g.Kluyveromyces lactis, Kluyveromyces marxianus), the Candida genus (e.g.Candida utilis, Candida cacaoi, Candida boidinii), the Geotrichum genus(e.g. Geotrichum fermentans), Hansenula polymorpha, Yarrowia lipolytica,or Schizosaccharomyces pombe. Preferred is the species Pichia pastoris.Examples for Pichia pastoris strains are X33, GS115, KM71, KM71H; andCBS7435.

In one embodiment, the eukaryotic cell is a fungal cell (e.g.Aspergillus (such as A. niger, A. fumigatus, A. orzyae, A. nidula),Acremonium (such as A. thermophilum), Chaetomium (such as C.thermophilum), Chrysosporium (such as C. thermophile), Cordyceps (suchas C. militaris), Corynascus, Ctenomyces, Fusarium (such as F.oxysporum), Glomerella (such as G. graminicola), Hypocrea (such as H.jecorina), Magnaporthe (such as M. orzyae), Myceliophthora (such as M.thermophile), Nectria (such as N. heamatococca), Neurospora (such as N.crassa), Penicillium, Sporotrichum (such as S. thermophile), Thielavia(such as T. terrestris, T. heterothallica), Trichoderma (such as T.reesei), or Verticillium (such as V. dahlia)).

In one embodiment, the eukaryotic cell is an insect cell (e.g., Sf9,Mimic™ Sf9, Sf21, High Five™ (BT1-TN-5B1-4), or BT1-Ea88 cells), analgae cell (e.g., of the genus Amphora, Bacillariophyceae, Dunaliella,Chlorella, Chlamydomonas, Cyanophyta (cyanobacteria), Nannochloropsis,Spirulina, or Ochromonas), or a plant cell (e.g., cells frommonocotyledonous plants (e.g., maize, rice, wheat, or Setaria), or froma dicotyledonous plants (e.g., cassava, potato, soybean, tomato,tobacco, alfalfa, Physcomitrella patens or Arabidopsis).

In one embodiment, the cell is a bacterial or prokaryotic cell.

In embodiments, the prokaryotic cell is a Gram-positive cells such asBacillus, Streptomyces Streptococcus, Staphylococcus or Lactobacillus.Bacillus that can be used is, e.g. the B. subtilis, B.amyloliquefaciens, B. licheniformis, B. natto, or B. megaterium. Inembodiments, the cell is B. subtilis, such as B. subtilis 3NA and B.subtilis 168. Bacillus is obtainable from, e.g., the Bacillus GeneticStock Center, Biological Sciences 556, 484 West 12^(th) Avenue, ColumbusOhio 43210-1214.

In one embodiment, the prokaryotic cell is a Gram-negative cell, such asSalmonella spp. or Escherichia coli, such as e.g., TG1, TG2, W3110, DH1,DHB4, DH5a, HMS 174, HMS174 (DE3), NM533, C600, HB101, JM109, MC4100,XL1-Blue and Origami, as well as those derived from E. coli B-strains,such as for example BL-21 or BL21 (DE3), all of which are commerciallyavailable.

Suitable host cells are commercially available, for example, fromculture collections such as the DSMZ (Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) or theAmerican Type Culture Collection (ATCC).

In embodiments, the cultured cells are used to produce proteins e.g.,antibodies, e.g., monoclonal antibodies, and/or recombinant proteins,for therapeutic use. In embodiments, the cultured cells producepeptides, amino acids, fatty acids or other useful biochemicalintermediates or metabolites. For example, in embodiments, moleculeshaving a molecular weight of about 4000 daltons to greater than about140,000 daltons can be produced. In embodiments, these molecules canhave a range of complexity and can include posttranslationalmodifications including glycosylation.

In embodiments, the protein is, e.g., BOTOX, Myobloc, Neurobloc, Dysport(or other serotypes of botulinum neurotoxins), alglucosidase alpha,daptomycin, YH-16, choriogonadotropin alpha, filgrastim, cetrorelix,interleukin-2, aldesleukin, teceleulin, denileukin diftitox, interferonalpha-n3 (injection), interferon alpha-nl, DL-8234, interferon, Suntory(gamma-1a), interferon gamma, thymosin alpha 1, tasonermin, DigiFab,ViperaTAb, EchiTAb, CroFab, nesiritide, abatacept, alefacept, Rebif,eptoterminalfa, teriparatide (osteoporosis), calcitonin injectable (bonedisease), calcitonin (nasal, osteoporosis), etanercept, hemoglobinglutamer 250 (bovine), drotrecogin alpha, collagenase, carperitide,recombinant human epidermal growth factor (topical gel, wound healing),DWP401, darbepoetin alpha, epoetin omega, epoetin beta, epoetin alpha,desirudin, lepirudin, bivalirudin, nonacog alpha, Mononine, eptacogalpha (activated), recombinant Factor VIII+VWF, Recombinate, recombinantFactor VIII, Factor VIII (recombinant), Alphnmate, octocog alpha, FactorVIII, palifermin,Indikinase, tenecteplase, alteplase, pamiteplase,reteplase, nateplase, monteplase, follitropin alpha, rFSH, hpFSH,micafungin, pegfilgrastim, lenograstim, nartograstim, sermorelin,glucagon, exenatide, pramlintide, iniglucerase, galsulfase, Leucotropin,molgramostim, triptorelin acetate, histrelin (subcutaneous implant,Hydron), deslorelin, histrelin, nafarelin, leuprolide sustained releasedepot (ATRIGEL), leuprolide implant (DUROS), goserelin, Eutropin, KP-102program, somatropin, mecasermin (growth failure), enlfavirtide,Org-33408, insulin glargine, insulin glulisine, insulin (inhaled),insulin lispro, insulin deternir, insulin (buccal, RapidMist),mecasermin rinfabate, anakinra, celmoleukin, 99 mTc-apcitide injection,myelopid, Betaseron, glatiramer acetate, Gepon, sargramostim,oprelvekin, human leukocyte-derived alpha interferons, Bilive, insulin(recombinant), recombinant human insulin, insulin aspart, mecasenin,Roferon-A, interferon-alpha 2, Alfaferone, interferon alfacon-1,interferon alpha, Avonex' recombinant human luteinizing hormone, dornasealpha, trafermin, ziconotide, taltirelin, diboterminalfa, atosiban,becaplermin, eptifibatide, Zemaira, CTC-111, Shanvac-B, HPV vaccine(quadrivalent), octreotide, lanreotide, ancestirn, agalsidase beta,agalsidase alpha, laronidase, prezatide copper acetate (topical gel),rasburicase, ranibizumab, Actimmune, PEG-Intron, Tricomin, recombinanthouse dust mite allergy desensitization injection, recombinant humanparathyroid hormone (PTH) 1-84 (sc, osteoporosis), epoetin delta,transgenic antithrombin III, Granditropin, Vitrase, recombinant insulin,interferon-alpha (oral lozenge), GEM-21S, vapreotide, idursulfase,omnapatrilat, recombinant serum albumin, certolizumab pegol,glucarpidase, human recombinant C1 esterase inhibitor (angioedema),lanoteplase, recombinant human growth hormone, enfuvirtide (needle-freeinjection, Biojector 2000), VGV-1, interferon (alpha), lucinactant,aviptadil (inhaled, pulmonary disease), icatibant, ecallantide,omiganan, Aurograb, pexigananacetate, ADI-PEG-20, LDI-200, degarelix,cintredelinbesudotox, Favld, MDX-1379, ISAtx-247, liraglutide,teriparatide (osteoporosis), tifacogin, AA4500, T4N5 liposome lotion,catumaxomab, DWP413, ART-123, Chrysalin, desmoteplase, amediplase,corifollitropinalpha, TH-9507, teduglutide, Diamyd, DWP-412, growthhormone (sustained release injection), recombinant G-CSF, insulin(inhaled, AIR), insulin (inhaled, Technosphere), insulin (inhaled,AERx), RGN-303, DiaPep277, interferon beta (hepatitis C viral infection(HCV)), interferon alpha-n3 (oral), belatacept, transdermal insulinpatches, AMG-531, MBP-8298, Xerecept, opebacan, AIDSVAX, GV-1001,LymphoScan, ranpirnase, Lipoxysan, lusupultide, MP52(beta-tricalciumphosphate carrier, bone regeneration), melanoma vaccine,sipuleucel-T, CTP-37, Insegia, vitespen, human thrombin (frozen,surgical bleeding), thrombin, TransMID, alfimeprase, Puricase,terlipressin (intravenous, hepatorenal syndrome), EUR-1008M, recombinantFGF-I (injectable, vascular disease), BDM-E, rotigaptide, ETC-216,P-113, MBI-594AN, duramycin (inhaled, cystic fibrosis), SCV-07, OPI-45,Endostatin, Angiostatin, ABT-510, Bowman Birk Inhibitor Concentrate,XMP-629, 99 mTc-Hynic-Annexin V, kahalalide F, CTCE-9908, teverelix(extended release), ozarelix, rornidepsin, BAY-504798, interleukin4,PRX-321, Pepscan, iboctadekin, rhlactoferrin, TRU-015, IL-21, ATN-161,cilengitide, Albuferon, Biphasix, IRX-2, omega interferon, PCK-3145,CAP-232, pasireotide, huN901-DMI, ovarian cancer immunotherapeuticvaccine, SB-249553, Oncovax-CL, OncoVax-P, BLP-25, CerVax-16,multi-epitope peptide melanoma vaccine (MART-1, gp100, tyrosinase),nemifitide, rAAT (inhaled), rAAT (dermatological), CGRP (inhaled,asthma), pegsunercept, thymosinbeta4, plitidepsin, GTP-200, ramoplanin,GRASPA, OBI-1, AC-100, salmon calcitonin (oral, eligen), calcitonin(oral, osteoporosis), examorelin, capromorelin, Cardeva, velafermin,131I-TM-601, KK-220, T-10, ularitide, depelestat, hematide, Chrysalin(topical), rNAPc2, recombinant Factor V111 (PEGylated liposomal), bFGF,PEGylated recombinant staphylokinase variant, V-10153, SonoLysisProlyse, NeuroVax, CZEN-002, islet cell neogenesis therapy, rGLP-1,BIM-51077, LY-548806, exenatide (controlled release, Medisorb),AVE-0010, GA-GCB, avorelin, ACM-9604, linaclotid eacetate, CETi-1,Hemospan, VAL (injectable), fast-acting insulin (injectable, Viadel),intranasal insulin, insulin (inhaled), insulin (oral, eligen),recombinant methionyl human leptin, pitrakinra subcutancous injection,eczema), pitrakinra (inhaled dry powder, asthma), Multikine, RG-1068,MM-093, NBI-6024, AT-001, PI-0824, Org-39141, Cpn10 (autoimmunediseases/inflammation), talactoferrin (topical), rEV-131 (ophthalmic),rEV-131 (respiratory disease), oral recombinant human insulin(diabetes), RPI-78M, oprelvekin (oral), CYT-99007 CTLA4-Ig, DTY-001,valategrast, interferon alpha-n3 (topical), IRX-3, RDP-58, Tauferon,bile salt stimulated lipase, Merispase, alaline phosphatase, EP-2104R,Melanotan-II, bremelanotide, ATL-104, recombinant human microplasmin,AX-200, SEMAX, ACV-1, Xen-2174, CJC-1008, dynorphin A, SI-6603, LABGHRH, AER-002, BGC-728, malaria vaccine (virosomes, PeviPRO), ALTU-135,parvovirus B19 vaccine, influenza vaccine (recombinant neuraminidase),malaria/HBV vaccine, anthrax vaccine, Vacc-5q, Vacc-4x, HIV vaccine(oral), HPV vaccine, Tat Toxoid, YSPSL, CHS-13340, PTH(1-34) liposomalcream (Novasome), Ostabolin-C, PTH analog (topical, psoriasis),MBRI-93.02, MTB72F vaccine (tuberculosis), MVA-Ag85A vaccine(tuberculosis), FARA04, BA-210, recombinant plague FIV vaccine, AG-702,OxSODrol, rBetV1, Der-pl/Der-p2/Der-p7 allergen-targeting vaccine (dustmite allergy), PR1 peptide antigen (leukemia), mutant ras vaccine,HPV-16 E7 lipopeptide vaccine, labyrinthin vaccine (adenocarcinoma), CMLvaccine, WT1-peptide vaccine (cancer), IDD-5, CDX-110, Pentrys, Norelin,CytoFab, P-9808, VT-111, icrocaptide, telbermin (dermatological,diabetic foot ulcer), rupintrivir, reticulose, rGRF, HA,alpha-galactosidase A, ACE-011, ALTU-140, CGX-1160, angiotensintherapeutic vaccine, D-4F, ETC-642, APP-018, rhMBL, SCV-07 (oral,tuberculosis), DRF-7295, ABT-828, ErbB2-specific immunotoxin(anticancer), DT3SSIL-3, TST-10088, PRO-1762, Combotox,cholecystokinin-B/gastrin-receptor binding peptides, 111In-hEGF, AE-37,trasnizumab-DM1, Antagonist G, IL-12 (recombinant), PM-02734, IMP-321,rhIGF-BP3, BLX-883, CUV-1647 (topical), L-19 basedradioimmunotherapeutics (cancer), Re-188-P-2045, AMG-386, DC/1540/KLHvaccine (cancer), VX-001, AVE-9633, AC-9301, NY-ESO-1 vaccine(peptides), NA17.A2 peptides, melanoma vaccine (pulsed antigentherapeutic), prostate cancer vaccine, CBP-501, recombinant humanlactoferrin (dry eye), FX-06, AP-214, WAP-8294A (injectable), ACP-HIP,SUN-11031, peptide YY [3-36] (obesity, intranasal), FGLL, atacicept,BR3-Fc, BN-003, BA-058, human parathyroid hormone 1-34 (nasal,osteoporosis), F-18-CCR1, AT-1100 (celiac disease/diabetes), JPD-003,PTH(7-34) liposomal cream (Novasome), duramycin (ophthalmic, dry eye),CAB-2, CTCE-0214, GlycoPEGylated erythropoietin, EPO-Fc, CNTO-528,AMG-114, JR-013, Factor XIII, aminocandin, PN-951, 716155, SUN-E7001,TH-0318, BAY-73-7977, teverelix (immediate release), EP-51216, hGH(controlled release, Biosphere), OGP-I, sifuvirtide, TV4710, ALG-889,Org-41259, rhCC10, F-991, thymopentin (pulmonary diseases), r(m)CRP,hepatoselective insulin, subalin, L19-IL-2 fusion protein, elafin,NMK-150, ALTU-139, EN-122004, rhTPO, thrombopoietin receptor agonist(thrombocytopenic disorders), AL-108, AL-208, nerve growth factorantagonists (pain), SLV-317, CGX-1007, INNO-105, oral teriparatide(eligen), GEM-OS1, AC-162352, PRX-302, LFn-p24 fusion vaccine(Therapore), EP-1043, S pneumoniae pediatric vaccine, malaria vaccine,Neisseria meningitidis Group B vaccine, neonatal group B streptococcalvaccine, anthrax vaccine, HCV vaccine (gpEl+gpE2+MF-59), otitis mediatherapy, HCV vaccine (core antigen+ISCOMATRIX), hPTH(1-34) (transdermal,ViaDerm), 768974, SYN-101, PGN-0052, aviscumnine, BIM-23190,tuberculosis vaccine, multi-epitope tyrosinase peptide, cancer vaccine,enkastim, APC-8024, GI-5005, ACC-001, TTS-CD3, vascular-targeted TNF(solid tumors), desmopressin (buccal controlled-release), onercept, andTP-9201.

In some embodiments, the polypeptide is adalimumab (HUMIRA), infliximab(REMICADE™), rituximab (RITUXAN™/MAB THERA™) etanercept (ENBREL™)bevacizumab (AVASTIN™), trastuzumab (HERCEPTIN™), pegrilgrastim(NEULASTA™), or any other suitable polypeptide including biosimilars andbiobetters.

Other suitable polypeptides are those listed below in Table 5, and inTable 1 of US2016/0097074.

TABLE 5 Protein Product Reference Listed Drug interferon gamma-1bActimmune ® alteplase; tissue plasminogen activator Activase ®/Cathflo ®Recombinant antihemophilic factor Advate human albumin Albutein ®Laronidase Aldurazyme ® Interferon alfa-N3, human leukocyte derivedAlferon N ® human antihemophilic factor Alphanate ® virus-filtered humancoagulation factor IX AlphaNine ® SD Alefacept; recombinant, dimericfusion protein Amevive ® LFA3-Ig Bivalirudin Angiomax ® darbepoetin alfaAranesp ™ Bevacizumab Avastin ™ interferon beta-1a; recombinant Avonex ®coagulation factor IX BeneFix ™ Interferon beta-1b Betaseron ®Tositumomab BEXXAR ® antihemophilic factor Bioclate ™ human growthhormone BioTropin ™ botulinum toxin type A BOTOX ® Alemtuzumab Campath ®acritumomab; technetium-99 labeled CEA-Scan ® alglucerase; modified formof beta- Ceredase ® glucocerebrosidase imiglucerase; recombinant form ofbeta- Cerezyme ® glucocerebrosidase crotalidae polyvalent immune Fab,ovine CroFab ™ digoxin immune fab [ovine] DigiFab ™ Rasburicase Elitek ®Etanercept ENBREL ® epoietin alfa Epogen ® Cetuximab Erbitux ™algasidase beta Fabrazyme ® Urofollitropin Fertinex ™ follitropin betaFollistim ™ Teriparatide FORTEO ® human somatropin GenoTropin ® GlucagonGlucaGen ® follitropin alfa Gonal-F ® antihemophilic factor Helixate ®Antihemophilic Factor; Factor XIII HEMOFIL adefovir dipivoxil Hepsera ™Trastuzumab Herceptin ® Insulin Humalog ® antihemophilic factor/vonWillebrand factor Humate-P ® complex-human Somatotropin Humatrope ®Adalimumab HUMIRA ™ human insulin Humulin ® recombinant humanhyaluronidase Hylenex ™ interferon alfacon-1 Infergen ® eptifibatideIntegrilin ™ alpha-interferon Intron A ® Palifermin Kepivance AnakinraKineret ™ antihemophilic factor Kogenate ® FS insulin glargine Lantus ®granulocyte macrophage colony-stimulating Leukine ®/Leukine ® Liquidfactor lutropin alfa for injection Luveris OspA lipoprotein LYMErix ™Ranibizumab LUCENTIS ® gemtuzumab ozogamicin Mylotarg ™ GalsulfaseNaglazyme ™ Nesiritide Natrecor ® Pegfilgrastim Neulasta ™ OprelvekinNeumega ® Filgrastim Neupogen ® Fanolesomab NeutroSpec ™ (formerlyLeuTech ®) somatropin [rDNA] Norditropin ®/Norditropin Nordiflex ®Mitoxantrone Novantrone ® insulin; zinc suspension; Novolin L ® insulin;isophane suspension Novolin N ® insulin, regular; Novolin R ® InsulinNovolin ® coagulation factor VIIa Novo Seven ® Somatropin Nutropin ®immunoglobulin intravenous Octagam ® PEG-L-asparaginase Oncaspar ®abatacept, fully human soluable fusion protein Orencia ™ muromomab-CD3Orthoclone OKT3 ® high-molecular weight hyaluronan Orthovisc ® hunanchorionic gonadotropin Ovidrel ® live attenuated BacillusCalmette-Guerin Pacis ® peginterferon alfa-2a Pegasys ® pegylatedversion of interferon alfa-2b PEG-Intron ™ Abarelix (injectablesuspension); Plenaxis ™ gonadotropin-releasing hormone antagonistepoietin alfa Procrit ® Aldesleukin Proleukin, IL-2 ® SomatremProtropin ® dornase alfa Pulmozyme ® Efalizumab; selective, reversibleT-cell blocker RAPTIVA ™ combination of ribavirin and alpha interferonRebetron ™ Interferon beta 1a Rebif ® antihemophilic factorRecombinate ® rAHF/ antihemophilic factor ReFacto ® Lepirudin Refludan ®Infliximab REMICADE ® Abciximab ReoPro ™ Reteplase Retavase ™ RituximaRituxan ™ interferon alfa-2^(a) Roferon-A ® Somatropin Saizen ®synthetic porcine secretin SecreFlo ™ Basiliximab Simulect ® EculizumabSOLIRIS ® Pegvisomant SOMAVERT ® Palivizumab; recombinantly produced,Synagis ™ humanized mAb thyrotropin alfa Thyroge ® Tenecteplase TNKase ™Natalizumab TYSABRI ® human immune globulin intravenous 5% andVenoglobulin-S ® 10% solutions interferon alfa-n1, lymphoblastoidWellferon ® drotrecogin alfa Xigris ™ Omalizumab; recombinantDNA-derived Xolair ® humanized monoclonal antibody targetingimmunoglobulin-E Daclizumab Zenapax ® ibriturnomab tiuxetan Zevalin ™Somatotropin Zorbtive ™ (Serostim ®)

In embodiments, the polypeptide is a hormone, blood clotting/coagulationfactor, cytokine/growth factor, antibody molelcule, fusion protein,protein vaccine, or peptide as shown in Table 3.

In embodiments, the protein is multispecific protein, e.g., a bispecificantibody as shown in Table 4.

EXAMPLES

The system and method of the present disclosure are further described indetail by reference to the following experimental examples. Theseexamples are provided for purposes of illustration only, and are notintended to be limiting unless otherwise specified. Thus, the disclosureshould in no way be construed as being limited to the followingexamples, but rather, should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentdisclosure and practice the claimed methods. The following workingexamples specifically point out various aspects of the presentdisclosure, and are not to be construed as limiting in any way theremainder of the disclosure.

Example 1 Processing Solution Components for Processing a BioproductAccording to the Disclosure

Tables 6-9 list the components, ingredient concentrations and bufferrequirements of a processing solution according to the disclosure forone biologic prepared in a bioreactor. Buffers A-L in Tables 7-9correspond to Buffers A-L identified in Table 6.

The bioprocess container can be used to contain the following buffers:Tris, Tris-Base, Tricine, HEPES, MOPS, PIPES, TAPS, bicine, BES, TES,cacodylate, MES, acetate, MKP, ADA, ACES, glycinamide, acetamidoglycine,acetic acid, citric acid, glycine, glycine glycinate, sodium phosphate,ethanol, hydrochloric acid, sodium hydroxide, guanidinium chloride,guanidine hydrochloride, sodium chloride, or a combination of any ofthese buffers or other buffers.

Tris is also known as tris(hydroxymethyl)aminomethane. HEPES is alsoknown as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. Tris-Baseis also known as 2-Amino-2-(hydroxymethyl)-1,3-propanediol, THAM, Trisbase, and Tris(hydroxymethyl)aminomethane, Trometamol. MOPS is alsoknown as 3-(N-morpholino)propanesulfonic acid. PIPES is also known aspiperazine-N,N′-bis(2-ethanesulfonic acid). TAPS is also known as[tris(hydroxymethyl)methylamino]propanesulfonic acid. BES is also knownas N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid,N,N-Bis(2-hydroxyethyl)taurine. TES is also known as2-[(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid,N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid, and TES freeacid. MES is also known as 2-(N-morpholino)ethanesulfonic acid. MKP isalso known as Monopotassium phosphate. ADA is also known asN-(2-Acetamido)iminodiacetic acid, N-(Carbamoylmethyl)iminodiaceticacid. ACES is also known as N-(2-Acetamido)-2-aminoethanesulfonic acid.

TABLE 6 Max Conc Number of Total Number Factor 2000 L Bags Volume of2000 L (capped at Volume After for Code Buffer (1X) bags 10X)Concentration Concentrate Buffer A 6M Gua-HCl 8,534 5 1 8534 9 Buffer B0.1M Acetic Acid, 8,534 5 1 8534 9 18% EtOH Buffer C 50 mM Glycine38,581 20 8 4823 5 Glycinate, 250 mM NaCl (pH 8.0) Buffer D 0.1MGlycine, 0.1M 14,935 8 10 1494 2 NaCl (pH 3.5) Buffer E 0.1M Citric AcidpH 6,401 4 10 640 1 2.1 Buffer F 18% Ethanol 800 1 1 800 1 Buffer G 1MHCl 30 1 1 30 1 Buffer H 1M Tris-Base 49 1 1 49 1 Buffer I 20 mM Na-19,127 10 10 1913 2 phosphate, 80 mM NaCl (pH 6.5) Buffer J 0.1M NaOH13,577 7 10 1358 2 Buffer K 20 mM Na- 3,556 2 1 3556 4 phosphate, 2MNaCl (pH 6.5) Buffer L 0.01M NaOH 2,546 2 10 255 1

TABLE 7 Total Mass Mass Buffer compo- Compo- Compo- nent 1 nent nentsComponent 1 (g/L) 1 (g) Buffer A Guanidine hydrochloride 573 4890168.19Buffer B Glacial acetic acid 6.50 55473.1121 Buffer C Glycine USP 7.20138891.1716 Buffer D Glycine USP 7.50 112013.0148 Buffer E Citric acidmonohydrate 20.79 133071.4616 Buffer F Ethanol absolute 145.00116013.4796 Buffer G Hydrochloric acid 2.0N 515.00 15602.87957 solutionBuffer H Tromethamine 117.57 5727.69772 Buffer I Sodium phosphatemonobasic 1.84 35193.05152 monohydrate Buffer J sodium hydroxide pellets4.00 54309.34052 Buffer K Sodium phosphate monobasic 0.51 1813.54405monohydrate Buffer L sodium hydroxide pellets 0.40 1018.300135

TABLE 8 Buffer Mass Total Mass Compo- component 2 Component nentsComponent 2 (g/L) 2 (g) Buffer A Buffer B Ethanol Absolute 1451237477.116 Buffer C Glycine sodium salt hydrate 0.28 5401.323339 BufferD Sodium Chloride 5.84 87220.80088 Buffer E Buffer F Buffer G Buffer HBuffer I Sodium phosphate dibasic 1.79 34236.71859 heptahydrate Buffer JSodium hydroxide 50% 5.22 70873.68938 solution Buffer K Sodium phosphatedibasic 3.97 14117.19584 heptahydrate Buffer L Sodium hydroxide 50%0.523 1331.427426 solution

TABLE 9 Total Mass Buffer Mass component 3 Component ComponentsComponent 3 (g/L) 3 (g) Buffer A Buffer B Buffer C Sodium chloride 29.1561352 Buffer D Hydrochloric acid as required Buffer E Buffer F Buffer GBuffer H Buffer I Sodium chloride 4.68 89512.76146 Buffer J Buffer KSodium chloride 108.22 384827 Buffer L

The disclosure will be further illustrated in the following claims.

What is claimed is:
 1. A system for delivering a processing solution,the system comprising: a bioprocess container containing a concentratedprocessing solution, the concentrated processing solution produced at afirst site; a processing area having at least one processing unit at asecond site different from the first site; and a pipe connecting thebioprocess container to the at least one processing unit, the pipehaving a valve to allow the concentrated processing solution to flowfrom the bioprocess container to the at least one processing unit tocombine the concentrated processing solution with a biopolymercontaining solution produced in the at least one processing unit,wherein the processing solution is at least one of a buffer and media.2. The system of claim 1, further comprising a controller connected tothe at least one processing unit, wherein the concentrated processingsolution is provided based on a determination by the controller that theprocessing solution is required when the determination is communicatedfrom the second site to the first site.
 3. The system of claim 1,wherein the system is configured to dilute the concentrated processingsolution prior to combining with the biopolymer containing solution. 4.The system of claim 1, wherein the processing solution is one of Tris,Tris-Base, Tricine, HEPES, MOPS, PIPES, TAPS, bicine, BES, TES,cacodylate, MES, acetate, MKP, ADA, ACES, glycinamide, acetamidoglycine,acetic acid, citric acid, glycine, glycine glycinate, sodium phosphate,ethanol, hydrochloric acid, sodium hydroxide, guanidinium chloride,guanidine hydrochloride, sodium chloride, and a combination of any ofthese.
 5. The system of claim 1, wherein the bioprocess containerincludes an inner layer for contacting the processing solution and anouter layer configured to support the inner layer.
 6. The system ofclaim 5, wherein the inner layer is polyethylene, and wherein the outerlayer is a blend of polyethylene, EVOH, nylon, and PVDC.
 7. The systemof claim 1, further comprising a controller configured to determine abioburden of the processing solution.
 8. The system of claim 1,comprising a dilution liquid supply, wherein the concentrated processingsolution from the bioprocess container and at least one of water andbuffer from the dilution liquid supply is added to an inlet of an inlineprocessing solution dilution system to result in a diluted processingsolution.
 9. The system of claim 1, the at least one processing unitfurther comprising a plurality of bioreactors.
 10. The system of claim1, further comprising a plurality of bioprocess containers.
 11. Thesystem of claim 8, wherein processing solution is a buffer.
 12. A methodfor delivering a processing solution, the method comprising providing aconcentrated processing solution in a bioprocess container, theconcentrated processing solution being produced at a first site; andcombining the concentrated processing solution with a biopolymercontaining solution produced in a bioreactor at a second site differentfrom the first site, wherein the processing solution is at least one ofa buffer and media.
 13. The method of claim 12, wherein the concentratedprocessing solution is provided based on a determination that theconcentrated processing solution is required and the determination iscommunicated from the second site to the first site.
 14. The method ofclaim 12, further comprising diluting the concentrated processingsolution prior to combining with the biopolymer containing solution. 15.The method of claim 12, wherein the processing solution is one of Tris,Tris-Base, Tricine, HEPES, MOPS, PIPES, TAPS, bicine, BES, TES,cacodylate, MES, acetate, MKP, ADA, ACES, glycinamide, acetamidoglycine,acetic acid, citric acid, glycine, glycine glycinate, sodium phosphate,ethanol, hydrochloric acid, sodium hydroxide, guanidinium chloride,guanidine hydrochloride, sodium chloride, and a combination of any ofthese.
 16. The method of claim 12, wherein the bioprocess containercomprises an inner layer for product contact and an outer layerconfigured to support the inner layer.
 17. The method of claim 16,wherein the inner layer is polyethylene, and wherein the outer layer isa blend of polyethylene, EVOH, nylon, and PVDC.
 18. The method of claim12, further comprising determining a bioburden of the processingsolution.
 19. The method of claim 12, further comprising addingconcentrated processing solution from the bioprocess container and atleast one of water and buffer from a dilution liquid supply to an inletof an inline processing solution dilution system to result in a dilutedprocessing solution.
 20. The method of claim 12, further comprisingtransporting the bioprocess container containing the processing solutionfrom the first site to the second site.
 21. A pharmaceutical productionfacility comprising: a buffer storage area capable of receiving at leastone bioprocess container configured to contain a concentrated processingsolution; a processing area having at least one processing unit; a wallseparating the buffer storage area from the processing area; and atleast one pipe having a first end in the buffer storage area and asecond end in the processing area, the first end configured to connectto at least one bioprocess container of the at least one bioprocesscontainer, and the second end configured to connect to at least oneprocessing unit of the at least one processing unit.
 22. Thepharmaceutical production facility of claim 21, wherein the at least onepipe has a valve.
 23. The pharmaceutical production facility of claim21, wherein the at least one bioprocess container includes an innerlayer for contacting the processing solution and an outer layerconfigured to support the inner layer.
 24. The pharmaceutical productionfacility of claim 21, further comprising a platform in the bufferstorage area, the platform being configured to support the at least onebioprocess container, the at least one bioprocess container including aplurality of bioprocess containers, the platform having a first levelconfigured to support at least one bioprocess container of the pluralityof bioprocess containers and a second level configured to support atleast one bioprocess container of the plurality of bioprocesscontainers, wherein the platform is configured to allow a user to placea bioprocess container of the plurality of bioprocess containers ontothe platform using a forklift, and the platform being configured toallow a user to remove the respective bioprocess container from theplatform using the forklift.
 25. The pharmaceutical production facilityof claim 24, further comprising a plurality of inlets spaced apart alongthe platform, each inlet being connected to the at least one pipe, andeach inlet being configured to be connected to an outlet of a bioprocesscontainer of the plurality of bioprocess containers.
 26. Thepharmaceutical production facility of claim 21, wherein the processingarea is configured as a cleanroom environment.